Formula of anti-freezing liquid for sperms of micropterus salmoides and odontobutis potamophila

Abstract

The invention belongs to the field of ultralow-temperature cryopreservation of fish sperms, and particularly relates to an anti-freezing liquid formula for sperms of micropterus salmoides and odontobutis potamophila. The anti-freezing liquid comprises an anti-freezing agent and a diluent, wherein the anti-freezing agent comprises at least one of methanol, ethylene glycol, glycerol and DMSO; the diluent comprises a diluent and a dispersoid, and the diluent comprises at least one of a Ren's solution and a Hank's solution; the dispersoid comprises at least one of sodium chloride, astragalus polysaccharide, apigenin, taurine, sodium pyruvate and crocin. The anti-freezing liquid can be used for cryopreservation of sperms of micropterus salmoides and odontobutis potamophila, the cryopreserved sperms are good in quality, high in activation rate, long in movement time and long in service life, and the anti-freezing liquid guarantees the quality of the cryopreserved sperms by means of maintaining stability of cell membranes in the cryopreservation process, reducing the freezing point of the cryopreserved sperms and the like.

Description

Technical Field

[0001] The invention belongs to the field of ultra-low temperature freezing and preservation of fish sperm, and in particular relates to an antifreeze solution formula for sperm of largemouth sea bass and river snakehead. Background Art

[0002] Largemouth bass, commonly known as California bass, is one of the important freshwater aquaculture fish in my country. However, in recent years, this species has shown signs of germplasm degradation, early sexual maturity, slowed growth, and susceptibility to disease. Therefore, it is necessary to protect its excellent germplasm resources.

[0003] The river Channa belongs to the order Perciformes and the family Channidae, and has high economic value. However, due to problems such as difficulty in squeezing out semen and asynchronous development of males and females, sperm cannot be fully utilized in seed production, affecting the efficiency of large-scale reproduction.

[0004] Therefore, the development of antifreeze formula for ultra-low temperature cryopreservation of sperm of largemouth black bass and river snakehead is of great significance to improving their artificial breeding technology and preserving excellent germplasm resources. Summary of the invention

[0005] In order to solve the above technical problems, the present invention specifically provides the following technical solutions: The invention aims to provide an antifreeze solution for sperms of largemouth bass and river snakehead.

[0006] The antifreeze solution of the invention is obtained by mixing an antifreeze agent and a diluent.

[0007] Preferably, the antifreeze agent comprises at least one of methanol, ethylene glycol, glycerol and DMSO.

[0008] Preferably, the diluent includes a diluent and a dispersant. The diluent includes at least one of Ringer's solution and Hank's solution, and the dispersant includes at least one of sodium chloride, astragalus polysaccharide, apigenin, taurine, sodium pyruvate, and crocin.

[0009] Preferably, the antifreeze solution contains 8 vol%-20 vol% of antifreeze agent.

[0010] More preferably, the antifreeze solution contains 8 vol%-12 vol% of antifreeze agent.

[0011] In one embodiment of the present invention, the antifreeze solution includes an antifreeze agent, a diluent and an additive.

[0012] Preferably, the antifreeze agent is methanol.

[0013] Preferably, the diluent comprises a diluent and a dispersant.

[0014] Preferably, the diluent of the diluent is Ringer's solution.

[0015] Preferably, the dispersed substances of the diluent are sodium chloride, astragalus polysaccharide, apigenin, taurine, sodium pyruvate, and crocin.

[0016] Preferably, the diluent is based on 1L, and when preparing, 0.01-0.3g sodium chloride, 0.1-1g astragalus polysaccharide, 0.01-0.05g apigenin, 0.1-1g taurine, 0.01-0.1mg sodium pyruvate and 1-5mg crocin are mixed, and then Ren's solution is added to make the volume to 1L.

[0017] Preferably, the additive includes ethylglucamine and ethyl 3-hydroxybutyrate.

[0018] Preferably, the antifreeze solution contains 1-5g of ethylglucamine and 1-2g of ethyl 3-hydroxybutyrate, based on 1L of antifreeze solution. Glucoethylglucamine and ethyl 3-hydroxybutyrate play a role in the cryopreservation of sperm of largemouth bass and river snakehead. Glucoethylglucamine and ethyl 3-hydroxybutyrate may reduce the freezing point of frozen semen by combining with water molecules, thereby reducing the generation of ice crystals; in addition, ethylglucamine and ethyl 3-hydroxybutyrate may also reduce the exposure time of sperm to methanol, thereby reducing the toxic effect of methanol on sperm, and then protecting sperm, thereby ensuring the quality of frozen sperm, so that frozen sperm has a better activation rate, longer movement time and longer life after thawing.

[0019] In one embodiment of the present invention, the additive further includes ursolic acid.

[0020] Preferably, the antifreeze solution contains 1-2 g of ursolic acid, based on 1 L of antifreeze solution. Ursolic acid plays a role in the cryopreservation of sperm of largemouth bass and river snakehead. Ursolic acid may interact with cell membranes to maintain the stability of cell membranes during cryopreservation, further protecting sperm, thereby ensuring the quality of frozen sperm, and making frozen sperm have a better activation rate, longer motility time and longer lifespan after thawing.

[0021] The present invention also provides a method for preserving fish sperm, comprising the following steps: The male fish is injected with hormones; then semen is collected; the semen is mixed with the antifreeze solution of the present invention to obtain frozen semen; the frozen semen is divided into straws; then the temperature is lowered step by step, and finally frozen in liquid nitrogen.

[0022] Preferably, the male fish comprises at least one of largemouth bass and river snakehead.

[0023] Preferably, the hormone includes at least one of chorionic gonadotropin and luteinizing releasing hormone, which can be used to promote sperm maturation.

[0024] Preferably, semen and antifreeze solution are mixed in a volume ratio of 1:1-5.

[0025] Preferably, the step-by-step cooling includes equilibrating the frozen semen at 2-6°C for 5-10 minutes, then transferring it to a cooling device with a liquid nitrogen level of 3-4 cm, leaving it for 3-5 minutes, and finally freezing it in liquid nitrogen.

[0026] Compared with the prior art, the present invention provides an antifreeze solution for sperm of largemouth bass and river sand snakehead, the antifreeze solution comprising an antifreeze agent and a diluent. The antifreeze agent of the antifreeze solution is methanol, and methanol of an appropriate concentration is conducive to the cryopreservation of sperm. The diluent of the antifreeze agent comprises a diluent and a dispersant, the diluent is Ren's solution, and the dispersant comprises sodium chloride, astragalus polysaccharide, apigenin, taurine, sodium pyruvate, and crocin. Dispersants such as sodium chloride, astragalus polysaccharide, apigenin, etc. play a role in the cryopreservation process of sperm and protect the cryopreservation of sperm. The present invention further adds additives to the antifreeze solution, and the additives include ethylglucamine, ethyl 3-hydroxybutyrate, and ursolic acid. The addition of additives ensures the quality of frozen sperm by maintaining the stability of cell membranes during the freezing process and lowering the freezing point of frozen semen, so that the frozen sperm has a better activation rate, longer movement time and longer lifespan after thawing, thereby improving the artificial breeding technology of largemouth black bass and river sand catfish, which is of great significance to the preservation of the excellent germplasm resources of largemouth black bass and river sand catfish. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Flowchart for semen cryopreservation. DETAILED DESCRIPTION

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] The experimental methods in the following examples, unless otherwise specified, are all conventional methods or conditions recommended by the manufacturer. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0030] The cryopreservation process of fish sperm is as follows Figure 1 As shown, the fish included largemouth bass and river snakehead, which were obtained from the Deqing Base of Zhejiang Freshwater Fisheries Research Institute. Male fish with well-developed gonads, good health, no injuries or diseases on the body surface and sexual maturity were selected as experimental materials.

[0031] Semen collection for largemouth bass involves the following steps: Human chorionic gonadotropin (HCG) and luteinizing hormone-releasing hormone (LRH-A2) were injected into the male fish 14 hours before semen collection to promote sperm maturation. Then the abdomen was directly squeezed for semen collection.

[0032] Semen collection of river Channa includes the following steps: 14 hours before semen collection, human chorionic gonadotropin (HCG) and luteinizing hormone-releasing hormone (LRH-A2) were injected into the male fish to promote sperm maturation. Then the fish abdomen was opened, the testes were taken out and quickly cut into pieces in a clean culture dish to avoid contact with water for semen collection.

[0033] Example 1: Ultra-low temperature cryopreservation of fish sperm The collected semen is placed in antifreeze to obtain frozen semen. The semen and antifreeze in the frozen semen are mixed at a volume ratio of 1:5, and then balanced at 4°C for 10 minutes, and then divided into straws, and the straws are placed flat on a cooling device for 5 minutes. The cooling device is located 4 cm above the liquid nitrogen surface, and then the straws are quickly transferred to liquid nitrogen for storage.

[0034] The antifreeze solution includes an antifreeze agent and a diluent. The antifreeze agent and the diluent are mixed at a volume ratio of 1:9 to obtain the antifreeze solution, which contains 10 vol% of the antifreeze agent.

[0035] The antifreeze agent is methanol. The diluent includes a diluent and a dispersant, the diluent is Ren's solution, and the dispersant includes sodium chloride, astragalus polysaccharide, apigenin, taurine, sodium pyruvate and crocin. When preparing the diluent, 0.3g sodium chloride, 1g astragalus polysaccharide, 0.05g apigenin, 1g taurine, 0.1mg sodium pyruvate, and 5mg crocin are mixed, and then Ren's solution is added and the volume is fixed to 1L.

[0036] Example 2: Ultra-low temperature cryopreservation of fish sperm The difference between Example 2 and Example 1 is that the diluent of the diluent of Example 2 is Hank's solution. When preparing the diluent, 0.3 g sodium chloride, 1 g astragalus polysaccharide, 0.05 g apigenin, 1 g taurine, 0.1 mg sodium pyruvate, and 5 mg crocin were mixed, and then Hank's solution was added and the volume was fixed to 1 L.

[0037] Example 3: Ultra-low temperature cryopreservation of fish sperm The difference between Example 3 and Example 2 is that the dispersed phase of the diluent of Example 3 is sodium chloride. When preparing the diluent, Hank's solution was added to 0.3 g of sodium chloride and the volume was fixed to 1 L.

[0038] Example 4: Ultra-low temperature cryopreservation of fish sperm The difference between Example 4 and Example 1 is that, in the antifreeze solution of Example 4, the antifreeze agent and the diluent are mixed at a volume ratio of 3:17 to obtain the antifreeze solution, and the antifreeze solution contains 15 vol% of the antifreeze agent.

[0039] Example 5: Ultra-low temperature cryopreservation of fish sperm The difference between Example 5 and Example 1 is that in the antifreeze solution of Example 5, the antifreeze agent and the diluent are mixed at a volume ratio of 2:8 to obtain the antifreeze solution, and the antifreeze solution contains 20 vol% of the antifreeze agent.

[0040] Example 6: Ultra-low temperature cryopreservation of fish sperm The difference between Example 6 and Example 1 is that the antifreeze solution of Example 6 further includes ethylglucamine and ethyl 3-hydroxybutyrate, wherein each 1L of the antifreeze solution contains 1g of ethylglucamine and 2g of ethyl 3-hydroxybutyrate.

[0041] Example 7: Ultra-low temperature cryopreservation of fish sperm The difference between Example 7 and Example 6 is that in Example 7, each 1 L of antifreeze solution contains 5 g of ethylglucamine and 2 g of ethyl 3-hydroxybutyrate.

[0042] Example 8: Ultra-low temperature cryopreservation of fish sperm The difference between Example 8 and Example 7 is that the antifreeze solution of Example 8 further includes ursolic acid, wherein each 1L of the antifreeze solution contains 1g of ursolic acid.

[0043] Example 9: Ultra-low temperature cryopreservation of fish sperm The difference between Example 9 and Example 8 is that in Example 9, each 1L of antifreeze solution contains 2g of ursolic acid.

[0044] Comparative Example 1: Ultra-low temperature cryopreservation of fish sperm The difference between Comparative Example 1 and Example 1 is that the antifreeze agent in Comparative Example 1 is DMSO.

[0045] Comparative Example 2: Ultra-low temperature cryopreservation of fish sperm The difference between Comparative Example 2 and Example 1 is that the antifreeze solution in Comparative Example 2 further includes ethylglucamine, wherein each 1L of the antifreeze solution contains 1g of ethylglucamine.

[0046] Comparative Example 3: Ultra-low temperature cryopreservation of fish sperm The difference between Comparative Example 3 and Example 1 is that the antifreeze solution in Comparative Example 3 also includes ethyl 3-hydroxybutyrate, wherein each 1L of the antifreeze solution contains 2g of ethyl 3-hydroxybutyrate.

[0047] Test Example 1: Sperm Activation Rate The activation rate refers to the percentage of motile sperm in the same field of view observed under a microscope after sperm activation to the total number of sperm.

[0048] The straw tubes containing sperm of largemouth bass or river chanca in Examples 1-9 and Comparative Examples 1-3 were quickly taken out of liquid nitrogen, immediately placed in a 40°C water bath and shaken to melt, the thawed frozen sperm was mixed with pure water at a volume ratio of 1:9, activated, and then the activation rate of the frozen sperm was detected under a microscope. In addition, before the largemouth bass or river chanca sperm was ultra-low temperature cryopreserved with liquid nitrogen, a portion of the frozen semen was aspirated and activated with pure water, and the activation rate of fresh sperm (fresh sperm) of largemouth bass or river chanca was detected under a microscope. The experiment was repeated 3 times.

[0049] The results are shown in Table 1. Compared with Comparative Example 1, Example 1 shows that methanol is more suitable for sperm antifreeze than DMSO. DMSO may be toxic to sperm, thereby reducing the activation rate of sperm; and compared with Examples 1, 4, and 5, it is shown that high concentrations of methanol may also be toxic to sperm, resulting in a decrease in sperm activation rate; and compared with Examples 1-3, it is shown that the diluent of Example 1 is more suitable for sperm antifreeze. Ren's solution is commonly used for ultra-low temperature cryopreservation of sperm of fish such as American shad and yellow catfish, while dispersoids such as astragalus polysaccharide and apigenin protect frozen sperm, resulting in a higher sperm activation rate.

[0050] Comparison between Example 1 and Comparative Examples 2 and 3 shows that adding ethylglucamine or ethyl 3-hydroxybutyrate alone to the antifreeze solution cannot significantly increase the activation rate of sperm; while comparison between Examples 1, 6, and 7 shows that adding ethylglucamine and ethyl 3-hydroxybutyrate to the antifreeze solution can significantly increase the activation rate of sperm, and ethylglucamine and ethyl 3-hydroxybutyrate may work synergistically to protect frozen sperm. Glucoethylglucamine and ethyl 3-hydroxybutyrate may reduce the freezing point of frozen semen by combining with water molecules, thereby reducing the formation of ice crystals; in addition, ethylglucamine and ethyl 3-hydroxybutyrate may also reduce the exposure time of sperm to methanol, thereby reducing the toxic effect of methanol on sperm, and then protecting sperm, thereby increasing the activation rate of sperm.

[0051] Comparison of Examples 7, 8, and 9 shows that further adding ursolic acid to the antifreeze solution can also improve the activation rate of sperm. Ursolic acid may interact with the cell membrane to maintain the stability of the cell membrane during the freezing process, further protecting the sperm, thereby improving the activation rate of sperm.

[0052] Table 1 Sperm activation rate

[0053] Test Example 2: Sperm Motility Time Sperm motility time refers to the time from when sperm is activated to when 90% of the sperm is trembling in place.

[0054] The straw tubes containing sperm of largemouth bass or river chanca in Examples 1-9 and Comparative Examples 1-3 were quickly taken out of liquid nitrogen, immediately placed in a 40°C water bath and shaken to melt, the thawed frozen semen was mixed with pure water at a volume ratio of 1:9, activated, and then the movement time of the frozen sperm was detected under a microscope. In addition, before the sperm of largemouth bass or river chanca was ultra-low temperature cryopreserved with liquid nitrogen, part of the frozen semen was aspirated and activated with pure water, and the movement time of fresh sperm (fresh sperm) of largemouth bass or river chanca was detected under a microscope. The experiment was repeated 3 times.

[0055] The results are shown in Table 2. Compared with Comparative Example 1, Example 1 shows that methanol is more suitable for sperm antifreeze than DMSO. DMSO may have a toxic effect on sperm, thereby reducing the motility time of sperm; and compared with Examples 1, 4, and 5, it is shown that high concentrations of methanol may also have a toxic effect on sperm, thereby reducing the motility time of sperm; and compared with Examples 1-3, it is shown that the diluent of Example 1 is more suitable for sperm antifreeze; Ren's solution is commonly used for ultra-low temperature cryopreservation of sperm of fish such as American shad and yellow catfish, while dispersed substances such as astragalus polysaccharide and apigenin protect frozen sperm, making the motility time of sperm longer.

[0056] Comparison between Example 1 and Comparative Examples 2 and 3 shows that adding ethylglucamine or ethyl 3-hydroxybutyrate alone to the antifreeze solution cannot significantly increase the motility time of sperm; while comparison between Examples 1, 6, and 7 shows that adding ethylglucamine and ethyl 3-hydroxybutyrate to the antifreeze solution can significantly increase the motility time of sperm, and ethylglucamine and ethyl 3-hydroxybutyrate may work synergistically to protect frozen sperm. Glucoethylglucamine and ethyl 3-hydroxybutyrate may reduce the freezing point of frozen semen by combining with water molecules, thereby reducing the formation of ice crystals; in addition, ethylglucamine and ethyl 3-hydroxybutyrate may also reduce the exposure time of sperm to methanol, thereby reducing the toxic effect of methanol on sperm, and then protecting sperm, thereby increasing the motility time of sperm.

[0057] Comparison of Examples 7, 8, and 9 shows that further adding ursolic acid to the antifreeze solution can also increase the motility time of sperm. Ursolic acid may interact with the cell membrane to maintain the stability of the cell membrane during the freezing process, further protecting the sperm, thereby increasing the motility time of sperm.

[0058] Table 2 Sperm movement time

[0059] Test Example 3: Sperm life span Lifespan refers to the time from when sperm is activated to when 90% of sperm stops moving.

[0060] The straw tubes containing sperm of largemouth bass or river chanca in Examples 1-9 and Comparative Examples 1-3 were quickly taken out of liquid nitrogen, immediately placed in a 40°C water bath and shaken to melt, the thawed frozen semen was mixed with pure water at a volume ratio of 1:9, activated, and then the lifespan of the frozen sperm was detected under a microscope. In addition, before the sperm of largemouth bass or river chanca was ultra-low temperature cryopreserved with liquid nitrogen, part of the frozen semen was taken and activated with pure water, and the lifespan of fresh sperm (fresh sperm) of largemouth bass or river chanca was detected under a microscope. The experiment was repeated 3 times.

[0061] The results are shown in Table 3. Compared with Comparative Example 1, Example 1 shows that methanol is more suitable for sperm antifreeze than DMSO. DMSO may be toxic to sperm, thereby reducing the life of sperm; while compared with Examples 1, 4, and 5, it is shown that high concentrations of methanol may also be toxic to sperm, thereby reducing the life of sperm; and compared with Examples 1-3, it is shown that the diluent of Example 1 is more suitable for sperm antifreeze; Ren's solution is commonly used for ultra-low temperature cryopreservation of sperm of fish such as American shad and yellow catfish, while additives such as astragalus polysaccharide and apigenin protect the frozen sperm, thereby extending the life of sperm.

[0062] Comparison between Example 1 and Comparative Examples 2 and 3 shows that adding ethylglucamine or ethyl 3-hydroxybutyrate alone to the antifreeze solution cannot significantly increase the lifespan of sperm; while comparison between Examples 1, 6, and 7 shows that adding ethylglucamine and ethyl 3-hydroxybutyrate to the antifreeze solution can significantly increase the lifespan of sperm, and ethylglucamine and ethyl 3-hydroxybutyrate may work synergistically to protect frozen sperm. Glucoethylglucamine and ethyl 3-hydroxybutyrate may reduce the freezing point of frozen semen by combining with water molecules, thereby reducing the formation of ice crystals; in addition, ethylglucamine and ethyl 3-hydroxybutyrate may also reduce the exposure time of sperm to methanol, thereby reducing the toxic effect of methanol on sperm, and then protecting sperm, thereby increasing the lifespan of sperm.

[0063] Comparison of Examples 7, 8, and 9 shows that further adding ursolic acid to the antifreeze solution can also increase the life span of sperm. Ursolic acid may interact with the cell membrane to maintain the stability of the cell membrane during the freezing process, further protecting the sperm, thereby increasing the life span of the sperm.

[0064] Table 3 Sperm life span

[0065] The embodiments and / or implementation methods described above are only used to illustrate the preferred embodiments and / or implementation methods for realizing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any form. Any technical personnel in this field may make slight changes or modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as technologies or embodiments that are essentially the same as the present invention.

Claims

1. An antifreeze solution, comprising an antifreeze agent and a diluent, characterized in that: The antifreeze agent includes at least one of methanol, ethylene glycol, glycerol and DMSO, the diluent includes a diluent and a dispersant, the diluent includes at least one of Ringer's solution and Hank's solution, and the dispersant includes at least one of sodium chloride, astragalus polysaccharide, apigenin, taurine, sodium pyruvate and crocin.

2. The antifreeze solution according to claim 1, characterized in that: The antifreeze solution further comprises an additive, wherein the additive comprises ethylglucamine and ethyl 3-hydroxybutyrate.

3. The antifreeze solution according to claim 1, characterized in that: The antifreeze solution contains 8 vol%-20 vol% of antifreeze agent.

4. The antifreeze solution according to claim 1, characterized in that: The diluent is Ringer's solution, and the dispersant is sodium chloride, astragalus polysaccharide, apigenin, taurine, sodium pyruvate, and crocin.

5. The antifreeze solution according to claim 2, characterized in that: The antifreeze solution comprises 1-5 g of ethylglucamine and 1-2 g of ethyl 3-hydroxybutyrate, based on 1 L of antifreeze solution.

6. The antifreeze solution according to claim 4, characterized in that: The diluent is calculated based on 1L; when preparing, 0.01-0.3g sodium chloride, 0.1-1g astragalus polysaccharide, 0.01-0.05g apigenin, 0.1-1g taurine, 0.01-0.1mg sodium pyruvate and 1-5mg crocin are mixed, and then Ren's solution is added to make the volume to 1L.

7. A method for preserving fish sperm, comprising the following steps: The male fish is injected with hormones; then semen is collected; the semen is mixed with the antifreeze solution of claim 1 to obtain frozen semen; the frozen semen is divided into straws; then the temperature is lowered step by step, and finally frozen in liquid nitrogen.

8. The storage method according to claim 7, characterized in that: The male fish includes at least one of a largemouth bass or a river snakehead.

9. The storage method according to claim 7, characterized in that: The hormone includes at least one of chorionic gonadotropin and luteinizing releasing hormone, which can be used to promote sperm maturation.

10. The storage method according to claim 7, characterized in that: The semen and antifreeze solution are mixed in a volume ratio of 1:1-5.

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