CN117256604B - Dolphin semen cryopreservation agent and semen cryopreservation method - Google Patents
Dolphin semen cryopreservation agent and semen cryopreservation method Download PDFInfo
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- CN117256604B CN117256604B CN202311570053.2A CN202311570053A CN117256604B CN 117256604 B CN117256604 B CN 117256604B CN 202311570053 A CN202311570053 A CN 202311570053A CN 117256604 B CN117256604 B CN 117256604B
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- 238000005138 cryopreservation Methods 0.000 title claims abstract description 31
- 238000000034 method Methods 0.000 title claims abstract description 19
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 89
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- 239000007788 liquid Substances 0.000 claims abstract description 67
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 claims abstract description 36
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- 210000002969 egg yolk Anatomy 0.000 claims abstract description 18
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 claims abstract description 18
- 239000001509 sodium citrate Substances 0.000 claims abstract description 18
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims abstract description 18
- 229960005322 streptomycin Drugs 0.000 claims abstract description 18
- 238000004321 preservation Methods 0.000 claims abstract description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 11
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims abstract description 9
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 9
- 108010024636 Glutathione Proteins 0.000 claims abstract description 9
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims abstract description 9
- 229930182555 Penicillin Natural products 0.000 claims abstract description 9
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims abstract description 9
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims abstract description 9
- 239000007983 Tris buffer Substances 0.000 claims abstract description 9
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims abstract description 9
- 239000008367 deionised water Substances 0.000 claims abstract description 9
- 229910021641 deionized water Inorganic materials 0.000 claims abstract description 9
- 239000008103 glucose Substances 0.000 claims abstract description 9
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- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 claims abstract description 6
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims abstract description 6
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- 229960001790 sodium citrate Drugs 0.000 claims abstract description 4
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- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 4
- 230000002338 cryopreservative effect Effects 0.000 claims description 2
- 210000004027 cell Anatomy 0.000 abstract description 17
- 230000004083 survival effect Effects 0.000 abstract description 12
- 230000007774 longterm Effects 0.000 abstract description 9
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Classifications
-
- 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
-
- 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
- A01N1/0215—Disinfecting agents, e.g. antimicrobials for preserving living parts
-
- 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
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a wide kiss dolphin semen cryopreservation agent and a semen cryopreservation method, and belongs to the technical field of dolphin semen cryopreservation. The wide kistrodon semen cryopreservation agent comprises a reagent I and a reagent II; wherein, the reagent I comprises the following components: tris, sodium citrate, streptomycin, egg yolk liquid and deionized water; reagent II comprises the following components: glucose, lactose, trehalose, sodium citrate, glutathione, penicillin, streptomycin, egg yolk liquid, deionized water and glycerol. The freeze preservative for the wide dolphin semen realizes long-term effective preservation of the wide dolphin sperm cells, and has high sperm survival rate.
Description
Technical Field
The invention belongs to the technical field of dolphin semen cryopreservation, and particularly relates to a wide kiss dolphin semen cryopreservation agent and a semen cryopreservation method.
Background
With the effects of global warming and human activity, the marine ecosystem is undergoing significant changes. These changes adversely affect the survival and proliferation of marine organisms such as dolphins. Thus, there is an increasing need to protect dolphin species, and semen cryopreservation is one of the important means to protect the genetic resources of species and promote reproduction. Genetic breeding of dolphin is one of important means for protecting dolphin species, and by freezing and preserving dolphin semen, genetic resources of rare dolphin species can be protected and utilized, irreversible loss of the genetic resources is avoided, and important experimental materials can be provided for the research of reproduction physiology and genomics of dolphin.
Since semen collection of terrestrial animals is relatively easy, semen cryopreservation technology is relatively mature in the field of terrestrial animal research, but semen cryopreservation technology of marine mammals is lacking. Moreover, conventional semen cryopreservors are not suitable for whale because the osmotic pressure of whale cells is far from that of terrestrial animals.
Therefore, how to provide a semen cryopreservation agent and a semen cryopreservation method suitable for dolphin to promote the protection of rare dolphin species is a technical problem to be solved currently.
Disclosure of Invention
Aiming at the technical problems, the invention provides the wide kistrodon semen cryopreservation agent and the semen cryopreservation method, which realize long-term effective preservation of wide kistrodon sperm cells and have high sperm survival rate.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the invention provides a dolphin semen cryopreservation agent, which comprises a reagent I and a reagent II; wherein, the reagent I comprises the following components: tris, sodium citrate, streptomycin, egg yolk liquid and deionized water; reagent II comprises the following components: glucose, lactose, trehalose, sodium citrate, glutathione, penicillin, streptomycin, egg yolk liquid, deionized water and glycerol; in the reagent I, the concentration of Tris is 30.28 g/L, the concentration of sodium citrate is 16.75 g/L, the concentration of streptomycin is 0.5 g/L, and the volume percentage concentration of egg yolk liquid is 20%; in the reagent II, the concentration of glucose is 22 g/L, the concentration of lactose is 37 g/L, the concentration of trehalose is 8 g/L, the concentration of sodium citrate is 6 g/L, the concentration of glutathione is 0.77 g/L, the concentration of penicillin is 0.6 g/L, the concentration of streptomycin is 1 g/L, the volume percentage concentration of egg yolk liquid is 20%, and the volume percentage concentration of glycerol is 3%.
Preferably, the osmotic pressure of the reagent I and the reagent II is 345+/-5 Osm/kg.
The invention also provides a method for freezing and preserving the wide kistrodon semen, which adopts the wide kistrodon semen freezing preservative according to any one of the technical schemes to freeze and preserve the semen, and comprises the following steps:
preparing a reagent I, regulating the osmotic pressure of the reagent I to 345+/-5 Osm/kg, and diluting the wide dolphin semen by using the reagent I to obtain an isotonic sample liquid;
mixing other components except glycerol in the reagent II, regulating osmotic pressure to 345+/-5 Osm/kg to obtain preliminary cooling protection liquid, and preheating at 36 ℃; diluting the isotonic sample liquid by using the preheated preliminary cooling protection liquid according to the volume ratio of 1:1, and cooling to 4-5 ℃ to obtain a preliminary cooling sample liquid;
mixing all components in the reagent II, and regulating osmotic pressure to 345+/-5 Osm/kg to obtain a cryoprotectant, wherein the volume percentage concentration of glycerol in the cryoprotectant is twice that in the reagent II; and (3) pre-cooling the freezing protection liquid at 4 ℃, diluting the primarily cooled sample liquid with the pre-cooled freezing protection liquid according to the volume ratio of 1:1, transferring the sample liquid into a freezing tube, cooling to-4 ℃, and then putting the sample liquid into liquid nitrogen for freezing preservation.
Preferably, the osmotic pressure is regulated by adopting series sodium chloride solutions with different concentrations, wherein the mass fractions of the series sodium chloride solutions are respectively 0.3%, 0.5%, 0.7%, 0.9%, 1.1%, 1.3%, 1.5%, 1.7% and 1.9%.
Preferably, the sperm concentration is adjusted to 40X10 when the wide kistrodon semen is diluted with reagent I 7 Sperm/ml.
Compared with the prior art, the invention has the advantages and positive effects that:
1. in the wide kistrodon semen cryopreservation agent provided by the invention, the reagent I is used as an intermediate buffer solution for diluting and transferring a wide kistrodon semen sample, and the reagent II is used for protecting in the freezing process; wherein, glucose, lactose and trehalose are used for providing energy, reducing the self-metabolism consumption of sperm cells and ensuring the metabolism consumption required by long-term preservation; sodium citrate is used to inhibit sperm cell motility; the glutathione is used for avoiding oxidative stress injury of spermatids and has a protective effect; penicillin and streptomycin are used for bacteriostasis; egg yolk liquid and glycerol can prevent damage to sperm cells due to freezing or water sublimation, and play a role in protection; the wide dolphin semen cryopreservation agent realizes long-term effective preservation of wide dolphin sperms through the cooperation of the components, and has high sperm survival rate;
2. the freeze preservative for the wide kistrodon semen provided by the invention can ensure that the osmotic pressure of the reagent I and the reagent II is consistent with that of the wide kistrodon semen, prevent the sperm from cracking or shrinking, and is beneficial to improving the survival rate of sperm cells;
3. according to the method for freezing and preserving wide kistrodon semen, the reagent I is used as a buffer diluent to obtain the isotonic sample liquid of the wide kistrodon semen, then two sections of cooling are adopted, the reagent II which does not contain glycerol is adopted in the first section to be mixed with the isotonic sample liquid of the wide kistrodon semen, the temperature is initially reduced to 4-5 ℃, the reagent II which contains glycerol is adopted in the second section to be mixed with the reagent II which contains glycerol, the temperature is reduced to-4 ℃, and finally the mixture is put into liquid nitrogen for freezing and preserving, so that the damage to spermatids can be reduced to the greatest extent, the survival rate of the spermatids is ensured, and the long-term effective preservation of the wide kistrodon spermatids is realized.
Drawings
Fig. 1 is a photograph of a sperm cell of a wide kistrodon stained with hematoxylin-eosin, wherein a is a photograph of viable sperm and B is a photograph of dead sperm, provided in example 1 of the present invention.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The embodiment of the invention provides a wide dolphin semen cryopreservation agent, which comprises a reagent I and a reagent II; wherein, the reagent I comprises the following components: tris, sodium citrate, streptomycin, egg yolk liquid and deionized water; reagent II comprises the following components: glucose, lactose, trehalose, sodium citrate, glutathione, penicillin, streptomycin, egg yolk liquid, deionized water and glycerol; in the reagent I, the concentration of Tris is 30.28 g/L, the concentration of sodium citrate is 16.75 g/L, the concentration of streptomycin is 0.5 g/L, and the volume percentage concentration of egg yolk liquid is 20%; in the reagent II, the concentration of glucose is 22 g/L, the concentration of lactose is 37 g/L, the concentration of trehalose is 8 g/L, the concentration of sodium citrate is 6 g/L, the concentration of glutathione is 0.77 g/L, the concentration of penicillin is 0.6 g/L, the concentration of streptomycin is 1 g/L, the volume percentage concentration of egg yolk liquid is 20%, and the volume percentage concentration of glycerol is 3%.
In the wide kistrodon semen cryopreservative, the reagent I is used as an intermediate buffer solution for diluting and transferring the wide kistrodon semen sample, and the reagent II is used for protecting in the freezing process; wherein, glucose, lactose and trehalose are used for providing energy, reducing the self-metabolism consumption of sperm cells and ensuring the metabolism consumption required by long-term preservation; sodium citrate is used to inhibit sperm cell motility; the glutathione is used for avoiding oxidative stress injury of spermatids and has a protective effect; penicillin and streptomycin are used for bacteriostasis; egg yolk liquid and glycerol can prevent damage to sperm cells due to freezing or sublimation of water, and protect sperm cells. The wide kistrodon semen cryopreservation agent realizes long-term effective preservation of wide kistrodon sperm cells through the cooperation of the components, and has high sperm survival rate. Meanwhile, the wide kiss dolphin semen cryopreservation agent limits the optimal concentration of each component in the reagent I and the reagent II, can obtain the optimal preservation effect, and the survival rate of sperm cells after half a year can reach more than 30%.
In a preferred embodiment, the osmotic pressure of both agent I and agent II is 345.+ -.5 Osm/kg. The preferred embodiment limits the osmotic pressure of the reagent I and the reagent II, can ensure that the osmotic pressure of the reagent I and the reagent II is consistent with the osmotic pressure of the wide dolphin semen, prevents the sperm from expanding or shrinking, and is beneficial to improving the survival rate of sperm cells.
The embodiment of the invention also provides a method for freezing and preserving the wide kistrodon semen, which adopts the wide kistrodon semen freezing and preserving agent to freeze and preserve the semen, and comprises the following steps:
(1) Preparing a reagent I, regulating the osmotic pressure of the reagent I to 345+/-5 Osm/kg, and diluting the wide dolphin semen by using the reagent I to obtain an isotonic sample liquid;
(2) Mixing other components except glycerol in the reagent II, regulating osmotic pressure to 345+/-5 Osm/kg to obtain preliminary cooling protection liquid, and preheating at 36 ℃; diluting the isotonic sample liquid by using the preheated preliminary cooling protection liquid according to the volume ratio of 1:1, and cooling to 4-5 ℃ to obtain a preliminary cooling sample liquid;
(3) Mixing all components in the reagent II, and regulating osmotic pressure to 345+/-5 Osm/kg to obtain a cryoprotectant, wherein the volume percentage concentration of glycerol in the cryoprotectant is twice that in the reagent II; and (3) pre-cooling the freezing protection liquid at 4 ℃, diluting the primarily cooled sample liquid with the pre-cooled freezing protection liquid according to the volume ratio of 1:1, transferring the sample liquid into a freezing tube, cooling to-4 ℃, and then putting the sample liquid into liquid nitrogen for freezing preservation.
In the above-mentioned method for cryopreserving the semen of Dolphin, glycerol has cytotoxicity, so that it can quickly permeate into the cell at a high temperature (for example, at room temperature or above), thereby generating toxicity to the sperm cell and causing sperm death, and at a low temperature, the permeation rate is low. Therefore, the invention adopts two sections of cooling, firstly adopts the reagent II which does not contain glycerol to mix with the isotonic sample liquid of the dolphin body, and then carries out preliminary cooling to 4-5 ℃, then mixes with the reagent II which contains glycerol, cools to-4 ℃, and finally enters into liquid nitrogen for freezing preservation.
According to the method for freezing and preserving the wide kistrodon semen, the reagent I is used as the buffer diluent to obtain the isotonic sample liquid of the wide kistrodon semen, then two sections of cooling are adopted, the reagent II which does not contain glycerol is adopted in the first section to be mixed with the isotonic sample liquid of the wide kistrodon semen, the temperature is initially lowered to 4-5 ℃, the reagent II which contains glycerol is mixed in the second section to be lowered to-4 ℃, and finally the mixture is put into liquid nitrogen for freezing and preserving, so that the damage to spermatids can be reduced to the greatest extent, the survival rate of the spermatids is guaranteed, and long-term effective preservation of the wide kistrodon spermatids is realized.
In a preferred embodiment, the osmotic pressure adjustment is performed by using different concentrations of the series of sodium chloride solutions, wherein the mass fractions of the series of sodium chloride solutions are respectively 0.3%, 0.5%, 0.7%, 0.9%, 1.1%, 1.3%, 1.5%, 1.7% and 1.9%, and the osmotic pressures of the series of sodium chloride solutions are shown in table 1.
Table 1 osmotic pressure of sodium chloride solutions
The specific steps of osmotic pressure regulation by adopting the series of sodium chloride solutions are as follows: when the osmotic pressure of the reagent (reagent I or reagent II) is lower than 340 Osm/kg, adding sodium chloride solution with osmotic pressure higher than 340 Osm/kg to adjust the osmotic pressure of the reagent, wherein when the difference is large, the sodium chloride solution with high osmotic pressure can be used for adjustment, and when the reagent is adjusted to be close to 340 Osm/kg, sodium chloride solution with osmotic pressure close to 340 Osm/kg is used for adjustment, so that the adding amount of the sodium chloride solution is reduced as much as possible; when the osmotic pressure of the reagent is higher than 350 Osm/kg, adding sodium chloride solution with the osmotic pressure lower than 340 Osm/kg to adjust the osmotic pressure of the reagent, wherein when the difference is large, the sodium chloride solution with the lower osmotic pressure can be used for adjusting, and when the reagent is adjusted to be close to 350 Osm/kg, the sodium chloride solution with the osmotic pressure close to 340 Osm/kg is used for adjusting, so that the adding amount of the sodium chloride solution is reduced as much as possible.
In a preferred embodiment, the sperm concentration is adjusted to 40X10 when the wide kistrodon semen is diluted with reagent I 7 Sperm/ml, at which concentration the final sperm concentration can be controlled at the most appropriate concentration after two further volumetric dilutions with reagent II.
In order to more clearly and in detail describe the wide kiss dolphin semen cryopreservation agent and the semen cryopreservation method provided by the embodiments of the present invention, the following description will be made with reference to specific embodiments.
Example 1
A dolphin semen cryopreservation agent comprises a reagent I and a reagent II; wherein, the reagent I comprises the following components: tris, sodium citrate, streptomycin, egg yolk liquid and deionized water, wherein the concentration of Tris is 30.28 g/L, the concentration of sodium citrate is 16.75 g/L, the concentration of streptomycin is 0.5 g/L, and the volume percentage concentration of egg yolk liquid is 20%; the components of reagent II and the amounts of the components are shown in Table 2.
The semen cryopreservation method adopting the wide kiss dolphin semen cryopreservation agent pair comprises the following steps:
(1) Preparation of reagent I and adjustment of osmotic pressure to 345.+ -.5 Osm/kg with a series of sodium chloride solutions of different concentrations (see Table 1);
(2) Transferring 250g wide kistrodon semen sample into centrifuge tube, centrifuging at 5000 rpm for 5 min, removing supernatant, diluting wide kistrodon semen with reagent I, counting with counter plate, and regulating sperm concentration to 40×10 7 Sperm/ml to obtain isotonic sample liquid;
(3) According to the components and the proportion of the reagent II in the table 2, mixing the components except glycerol in the reagent II, regulating the osmotic pressure to 345+/-5 Osm/kg by using a series of sodium chloride solutions with different concentrations (see table 1), obtaining a preliminary cooling protection solution, and preheating at 36 ℃;
(4) Diluting the isotonic sample liquid with the preheated preliminary cooling protection liquid according to the volume ratio of 1:1 within 5 min, gently vibrating the test tube to enable the sperms to suspend and mix uniformly, and placing 1.5 h at 5 ℃ to cool to 5 ℃ to obtain the preliminary cooling sample liquid;
(5) Mixing all the components in the reagent II, wherein the dosage of glycerol is twice that of the reagent II in table 2, regulating osmotic pressure to 345+/-5 Osm/kg by using a series of sodium chloride solutions with different concentrations (see table 1) to obtain a cryoprotectant, and pre-cooling the cryoprotectant at 4 ℃;
(6) Diluting the primarily cooled sample liquid with precooled cryoprotectant liquid according to the volume ratio of 1:1, keeping for 10min at 5 ℃, and transferring into a cryopreservation tube;
(7) Placing the freezing tube on a shelf 4-5cm above liquid nitrogen, standing for 10min to cool the freezing tube to-4deg.C, and freezing in liquid nitrogen.
TABLE 2 Components and amounts of the respective components of reagent II employed in example 1 and comparative examples 1 to 7
Comparative examples 1 to 7 differ from example 1 in the composition of reagent II, see in particular Table 2.
After half a year, the frozen wide kistrodon semen of example 1 and comparative examples 1-7 was thawed by the following method: and taking 10 mu L of frozen wide dolphin semen, and placing the wide dolphin semen in a sperm chamber preheated at 36 ℃ for 0.5h. After thawing, sperm survival was assessed using a Qinghai homonymous semen analyzer and the results are shown in Table 3. The photograph of wide kistrodon sperm cells after hematoxylin-eosin staining under a microscope is shown in figure 1.
TABLE 3 sperm preservation results for example 1 and comparative examples 1-7
As can be seen from Table 3, the semen sample frozen and preserved by the semen freezing preservative of example 1 still has the sperm survival rate of more than 30% after thawing, realizes the long-term effective preservation of the sperm cells of the Dolphin, and is beneficial to promoting the protection work of rare Dolphin species.
Claims (4)
1. The dolphin semen cryopreservation agent is characterized by comprising a reagent I and a reagent II; wherein the reagent I comprises the following components: tris, sodium citrate, streptomycin, egg yolk liquid and deionized water; the reagent II comprises the following components: glucose, lactose, trehalose, sodium citrate, glutathione, penicillin, streptomycin, egg yolk liquid, deionized water and glycerol; in the reagent I, the concentration of Tris is 30.28 g/L, the concentration of sodium citrate is 16.75 g/L, the concentration of streptomycin is 0.5 g/L, and the volume percentage concentration of egg yolk liquid is 20%; in the reagent II, the concentration of glucose is 22 g/L, the concentration of lactose is 37 g/L, the concentration of trehalose is 8 g/L, the concentration of sodium citrate is 6 g/L, the concentration of glutathione is 0.77 g/L, the concentration of penicillin is 0.6 g/L, the concentration of streptomycin is 1 g/L, the volume percentage concentration of egg yolk liquid is 20%, and the volume percentage concentration of glycerol is 3%; the osmotic pressure of the reagent I and the osmotic pressure of the reagent II are 345+/-5 Osm/kg.
2. A method for cryopreserving broad kiss dolphin semen, characterized in that the broad kiss dolphin semen cryopreservative agent in claim 1 is adopted for cryopreserving semen, comprising the following steps:
preparing a reagent I, regulating the osmotic pressure of the reagent I to 345+/-5 Osm/kg, and diluting the wide dolphin semen by using the reagent I to obtain an isotonic sample liquid;
mixing other components except glycerol in the reagent II, regulating osmotic pressure to 345+/-5 Osm/kg to obtain preliminary cooling protection liquid, and preheating at 36 ℃; diluting the isotonic sample liquid by using the preheated preliminary cooling protection liquid according to the volume ratio of 1:1, and cooling to 4-5 ℃ to obtain a preliminary cooling sample liquid;
mixing all components in the reagent II, and regulating osmotic pressure to 345+/-5 Osm/kg to obtain a cryoprotectant, wherein the volume percentage concentration of glycerol in the cryoprotectant is twice that in the reagent II; and (3) pre-cooling the freezing protection liquid at 4 ℃, diluting the primarily cooled sample liquid with the pre-cooled freezing protection liquid according to the volume ratio of 1:1, transferring the sample liquid into a freezing tube, cooling to-4 ℃, and then putting the sample liquid into liquid nitrogen for freezing preservation.
3. The method for cryopreserving wide kistrodon semen according to claim 2, wherein the osmotic pressure is adjusted by using a series of sodium chloride solutions with different concentrations, wherein the mass fractions of the series of sodium chloride solutions are 0.3%, 0.5%, 0.7%, 0.9%, 1.1%, 1.3%, 1.5%, 1.7% and 1.9%, respectively.
4. The method for cryopreserving wide kistrodon semen according to claim 2, wherein the concentration of sperm is adjusted to 4x10 when the wide kistrodon semen is diluted with reagent I 8 Sperm/ml.
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