Cryopreservation method for sperms of acrossocheilus longifin

Abstract

The invention belongs to the technical field of biology, and particularly relates to an acrossocheilus fasciatus sperm cryopreservation method which comprises the following steps: constructing a simulated water solution for acrossocheilus fasciatus in-vitro fertilization, collecting fresh sperms, detecting the survival rate, preparing a mixed solution of the sperms and anti-freezing components, sub-packaging, cooling and freezing and the like. According to the method, simulated water quality for in-vitro fertilization of acrossocheilus longifin is constructed, the survival rate of sperms observed and detected in the environment is closer to the fertilization condition during thawing activation in the future, and then the sperms with the higher fertilization success rate after thawing are screened out; meanwhile, the constructed simulated water solution can be used as a part of a cryopreservation solution, so that the remaining anti-freezing components can be directly added into the screened sperms, a mixed solution of the sperms and the anti-freezing components can be rapidly prepared under the condition that the service life of the sperms is not remarkably shortened, and the mixed solution is subpackaged, cooled and frozen; the improved anti-freezing component contains trehalose, soybean phospholipid and tea polyphenol, so that the survival rate and activity of the acrossocheilus fasciatus sperms after cryopreservation can be effectively improved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a method for freezing and preserving sperm of long-finned glaucus. Background Art

[0002] The longfin rayfish (Acrossocheilus longipinnis), a member of the Cyprinidae family and genus Acrossocheilus, is primarily distributed in the upper and middle reaches of the Pearl River Basin, particularly in tributaries and lakes with good water quality. Due to its graceful appearance and docile nature, it is sometimes cultivated as an ornamental fish and is an important commercial fish in the Pearl River Basin. Within the Pearl River Basin, the longfin rayfish typically inhabits shallow waters with slow currents and lush vegetation. However, over the past few decades, due to accelerated urbanization, water pollution, and water conservancy project construction, the longfin rayfish has faced certain threats to its habitat and is listed as Vulnerable on the China Red List. It was not until the past two years that China has made initial progress in the artificial breeding of longfin rayfish, for example [Wang Yuesong et al., 2024. "Study on artificial reproduction and early development of longfin rayfish (Acrossocheilus longipinnis)", Southern Fisheries Science, 20(2):63-72.doi:10.12131 / 20230207)], but the relevant research is still limited to the experimental stage and has not reached the production scale. The acquisition of fertilized eggs still depends on collecting wild parents for artificial insemination. In addition, due to the limited number of wild longfin rayfish captured each time, the small amount of male fish semen and the asynchronous development of male and female gonads, it is difficult to obtain mature sperm and mature eggs at the same time, which seriously hinders the progress of artificial breeding technology research. Therefore, it is very necessary to freeze and preserve longfin rayfish sperm.

[0003] The research and exploration of long-term cryopreservation of fish sperm is not only of great significance for further understanding the basic life process, but also plays a role in protecting fishery resources and cultivating excellent species by cryopreserving sperm of important economic fish and endangered fish. Existing cryopreservation solutions for fish sperm usually contain balanced salt solution, antifreeze (glycerol, DMSO and methanol, etc.) and nutritional components (skim milk powder, egg yolk, protein and sugar, etc.). For example, [Wang Xiaoai et al., 2012. "Ultra-low temperature cryopreservation of soft-finned rayfish sperm", Zoological Research, 2012, Jun. 33(3): 283-289] D-15 diluent was used with MeOH and EG to form a cryopreservation solution. However, the maximum motility of sperm after thawing was (29.67±0.47)%, which could not reach the production application level. Experiments have found that existing sperm cryopreservation solutions cannot preserve longfin rayfish sperm well, so it is necessary to improve sperm cryopreservation solutions for longfin rayfish sperm.

[0004] Currently, a common method for testing the survival rate of fish sperm is to activate the sperm with pure water, then observe under a microscope and test using a sperm survival assessment system (CASA). However, the survival rate and fertilization rate of longfin rayfish sperm with high activity screened using this method are still low after thawing and recovery. It is speculated that this is because the fertilization environment during natural fertilization of longfin rayfish sperm is not pure water. The sperm screened by activation with pure water according to conventional methods are not the most active sperm or the sperm most adapted to the real fertilization environment. Therefore, the present invention screens longfin rayfish sperm by simulating the fertilization environment, and the solution simulating the fertilization environment can be used as part of the sperm cryopreservation solution to avoid adverse effects on the cryopreservation of longfin rayfish sperm. Summary of the Invention

[0005] To achieve the above objectives, the present invention constructs a simulated environment for in vitro fertilization of longfin guppies, screening out sperm with a higher success rate of fertilization after thawing. Simultaneously, based on the simulated aqueous solution for in vitro fertilization of longfin guppies, the cryopreservation solution components are improved for longfin guppies sperm, significantly improving the survival rate and activity of longfin guppies sperm after cryopreservation. The specific technical solution employed is: a method for cryopreserving longfin guppies sperm, comprising the following steps:

[0006] Step 1. Construct a simulated aqueous solution for in vitro fertilization of longfin ray: add the following components to pure water: 16 g / L sodium chloride, 0.80 g / L potassium chloride, 2 g / L L-glucose, and 85.5 g / L trehalose. The water temperature is 16-20°C.

[0007] Step 2. Fresh semen collection and survival rate testing: During the longfin ray breeding season, select healthy, sexually mature male fish, dry the area around the cloaca with a dry towel, gently press the abdomen, squeeze the semen into a culture dish, and quickly transfer it to ice; use a 10-μl pipette to draw 10 μl of semen into a 200-μl centrifuge tube; draw 1 μl of semen and add 60 μl of the simulated water solution prepared in step 1 to activate the sperm. After mixing, observe sperm motility under a microscope on a glass slide and test sperm survival using a sperm survival rate testing system. Select semen with a fresh sperm survival rate of more than 70% for cryopreservation;

[0008] Step 3. Prepare a mixture of semen and antifreeze components: Use a pipette to transfer the diluted semen to a 50 mL centrifuge tube and measure its volume. Then, add the following liquid containing the following components in a 1:1 ratio by volume to the centrifuge tube: 0.096 g / L disodium hydrogen phosphate, 0.70 g / L sodium bicarbonate, 0.12 g / L potassium dihydrogen phosphate, 0.38 g / L calcium chloride dihydrate, 0.20 g / L magnesium chloride hexahydrate, 0.22 g / L magnesium sulfate heptahydrate, 16.5 g / L soybean lecithin, 150 mL / L methanol, and 300 μg / mL tea polyphenols. Mix thoroughly by pipetting and dissolve.

[0009] Step 4. Aliquot: Place 240 μL of the mixture prepared in step 3 into each 2 mL cryovial. Once all aliquots are complete, store them on ice.

[0010] Step 5: Cooling and freezing: Refrigerate the cryovials in a refrigerator at 4°C for 30 minutes, freeze at -20°C for 10 minutes, and freeze at -80°C for 12 minutes, then store in liquid nitrogen.

[0011] Moreover, the soybean lecithin in step 3 is in powder form.

[0012] Furthermore, in step 5, the cells were cooled to -80°C using a cell gradient cooling box and then stored in liquid nitrogen.

[0013] Moreover, when the stored longfin bream sperm is heated and thawed, a 2 mL cryovial containing semen is taken out from liquid nitrogen; the cryovial is quickly inserted into a 40°C water bath; the semen is continuously shaken for about 30 seconds until the semen is in an ice-water mixed state and thawed; after thawing, the simulated aqueous solution constructed in step 1 is added to activate the sperm.

[0014] Compared with the existing technology, the beneficial effects of this technical solution are:

[0015] 1. Simulating water conditions for in vitro fertilization of longfin ray bream. The sperm survival rates observed and tested in this environment more closely resemble the conditions for future fertilization upon thawing and activation. Under these conditions, sperm with a survival rate of over 70% are directly screened and cryopreserved. Compared to existing methods that use pure water or other methods to activate sperm, this method eliminates sperm that are more active in pure water but less active under actual fertilization conditions, resulting in a higher success rate for post-thaw fertilization. It also eliminates sperm that only exhibit oscillatory motion, rather than spiraling motion, under fertilization conditions, further improving the success rate of post-thaw fertilization.

[0016] 2. After constructing the simulated water quality for in vitro fertilization of longfin rayfish to screen sperm, it is necessary to avoid conflicts with the components of the cryopreservation solution. Therefore, the present invention directly designs the simulated water quality solution for in vitro fertilization of longfin rayfish to be part of the cryopreservation solution. On this basis, the components of the cryopreservation solution are further improved according to the characteristics of longfin rayfish sperm. Therefore, the sperm screened by the simulated water quality for in vitro fertilization of longfin rayfish can be directly added with the remaining antifreeze components. Under the condition of not significantly reducing the lifespan of sperm, a mixed solution of semen and antifreeze components is quickly prepared, and the mixture is packaged, cooled and frozen.

[0017] 3、The sperm of the long-finned pygmy sunfish will produce a large amount of active oxygen due to respiration during the cooling balance stage before cryopreservation, which causes damage to the sperm plasma membrane and affects the cryopreservation effect of the sperm of the long-finned pygmy sunfish. In view of this situation, the inventors have conducted experiments with egg yolk, but found that the egg yolk will react with a protein coagulation enzyme in the sperm of the long-finned pygmy sunfish, which has an adverse effect on the survival of the sperm. Soybean phospholipid is a plant product suitable for replacing egg yolk and can be used to maintain the stability of the cell membrane structure and effectively protect the sperm cell structure. However, the sperm of the long-finned pygmy sunfish lacks the acrosome structure of mammalian sperm, and the cryopreservation solution for mammalian sperm cannot be directly applied to the cryopreservation of the sperm of the long-finned pygmy sunfish. The method improves the antifreeze component, and the soybean phospholipid can be applied to the cryopreservation solution for the sperm of the long-finned pygmy sunfish.

[0018] 4、Although the soybean phospholipid as a plant-derived membrane stabilizer can effectively maintain the integrity of the cell membrane through phospholipid bilayer reconstruction, the sperm of the long-finned pygmy sunfish lacks the acrosome structure of mammalian sperm, and the response mechanism of its membrane system to freezing damage is essentially different. Therefore, the method constructs a composite antifreeze system and finds that the introduction of tea polyphenol improves the survival rate and activity of the sperm of the long-finned pygmy sunfish after cryopreservation. It is speculated that the antioxidant properties of tea polyphenol can target the removal of hydroxyl radicals and superoxide anions generated during the cooling process, and in addition, tea polyphenol can form a membrane structure-oxidation defense synergistic protection mechanism with soybean phospholipid. In addition, the optimization of the methanol osmotic pressure buffer system and the ratio of trehalose glassification protection in the improved cryopreservation solution also helps to improve the survival rate and activity of the sperm of the long-finned pygmy sunfish after cryopreservation. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The sperm of the long-finned pygmy sunfish observed after thawing and recovery for Example 1 (the dark circles in the figure are surviving sperm, and the white ones are inactivated sperm);

[0020] Figure 2 The sperm of the long-finned pygmy sunfish observed after thawing and recovery for Comparative Example 1;

[0021] Figure 3 The sperm of the long-finned pygmy sunfish observed after thawing and recovery for Comparative Example 2; DETAILED DESCRIPTION

[0022] The present application will be described in detail below in combination with the drawings and examples, and the content of the present application is not limited to the following examples.

[0023] Example 1:

[0024] (1) Construct a simulated water solution for in vitro fertilization of longfin ray: add the following components to pure water: 16g / L sodium chloride, 0.80g / L potassium chloride, 2g / L D-glucose, 85.5g / L trehalose, and adjust the pH to 7.5 and the water temperature to 18℃.

[0025] (2) Fresh semen collection and survival rate detection: During the breeding season of longfin bream, select healthy male fish that are sexually mature, wipe the area around the cloaca with a dry towel, press the abdomen lightly, squeeze the semen into a culture dish and quickly transfer it to ice; use a 10-μl pipette to draw 10 μl of semen into a 200-μl centrifuge tube, draw 1 μl of semen and add 60 μl of the simulated water solution constructed in step 1 to activate the sperm, mix well, observe the sperm movement under a microscope on a glass slide, and use a sperm survival rate detection system to detect the sperm survival rate, and select semen with a fresh sperm survival rate of more than 70% for cryopreservation.

[0026] (3) Prepare a mixture of semen and antifreeze components: Use a pipette to transfer the diluted semen to a 50 mL centrifuge tube, measure its volume at the same time, and then add the following components into the centrifuge tube in a 1:1 ratio: 0.096 g / L disodium hydrogen phosphate, 0.70 g / L sodium bicarbonate, 0.12 g / L potassium dihydrogen phosphate, 0.38 g / L calcium chloride dihydrate, 0.20 g / L magnesium chloride hexahydrate, 0.22 g / L magnesium sulfate heptahydrate, 16.5 g / L soybean lecithin, 150 mL / L methanol, 300 μg / mL tea polyphenols, and mix thoroughly by pipetting to dissolve.

[0027] (4) Sperm preservation: Measure 240 μL of the mixed solution of semen and antifreeze solution prepared in step 3 into a 2 mL cryovial. After all the parts are divided, place them on ice for temporary storage; refrigerate the cryovial in a refrigerator at 4°C for 30 minutes, freeze at -20°C for 10 minutes, and freeze at -80°C for 12 minutes, and then store them in liquid nitrogen; or use a cell gradient cooling box to gradually cool the cryovial to -80°C and then store them in liquid nitrogen.

[0028] (5) Thawing and resuscitation: After one month of cryopreservation, remove 6 groups of 2 mL cryovials containing semen from liquid nitrogen and test their survival rates. Quickly insert them into a 40°C water bath and shake them continuously for about 30 seconds until the semen is in an ice-water mixture. Pipette 1 μL of thawed semen onto a glass slide and adjust the microscope to the appropriate focus. Add 10 μL of the simulated aqueous solution constructed in step 1 to activate the sperm. Observe the sperm motility under a microscope and use the sperm survival system (CASA) to test the sperm survival rate. The results of the sperm survival rate test of the 6 groups are shown in Table 1. The average survival rate after thawing and resuscitation reached 59.3±5.4%, which is at the production application level. Figure 1This is the observation of the longfin ray sperm after thawing and recovery in the example, where the sperm survival rate refers to the ratio of surviving sperm to the total number of sperm in the field of view.

[0029] Table 1 shows the survival rates of 6 groups of longfin bream sperm after thawing and recovery in Example

[0030]

[0031] (6) Fertilization success rate experiment: The thawed and revived semen in step 5 was combined with female fish eggs under fertilization conditions. The fertilization rate was observed and recorded after 3 hours. The survival rate of fry was observed and recorded after 3 weeks. The results of the fertilization success rate experiments of 6 groups are shown in Table 2, where the fertilization rate refers to the ratio of fertilized eggs to the total number of eggs, and the survival rate of fry refers to the ratio of hatched eggs to the total number of fertilized eggs.

[0032] Table 2 is the experimental results of the fertilization success rate of 6 groups of long-finned bream sperm in the embodiment

[0033] Example group number Fertilization rate (%) Fry survival rate (%) 1 79.25 78.9 2 84.6 87.2 3 75.3 82.3 4 81.7 80.1 5 76.8 85.4 6 79.1 79.8

[0034] Comparative Example 1:

[0035] (1) Preparation of antifreeze solution: Use a pipette to draw 7.5 mL of methanol into a 50 mL centrifuge tube; add Hank's solution standard solution diluent to 50 mL and add it to the above centrifuge tube; use a vortex shaker to mix thoroughly and dissolve.

[0036] (2) Sperm collection: During the breeding season of longfin rayfish, cast a net to catch longfin rayfish males, select healthy males that are sexually mature, wipe the area around the cloaca with a dry towel, gently press the abdomen, squeeze the semen into a culture dish, and quickly transfer it to ice. Use a 10-μl pipette to draw 10 μl of semen into a 200-μl centrifuge tube, add 50 μl of Hank's solution standard solution diluent, pipette and mix; draw 1 μl of the mixture onto a glass slide, adjust the microscope to the appropriate focus; add 10 μl of pure water diluent to activate the sperm, observe the sperm movement under a microscope, use the sperm survival rate detection system (CASA) to detect the sperm survival rate, and select semen with a fresh sperm survival rate of more than 70% for cryopreservation.

[0037] (3) Sperm preservation: Use a pipette to transfer the semen into a 50 mL centrifuge tube and measure its volume at the same time; add antifreeze solution at a ratio of 1:1 and mix thoroughly by pipetting; measure 240 μL of the semen antifreeze solution mixture into a 2 mL cryovial, and after all the parts are divided, place them on ice for temporary storage; place the 2 mL cryovial containing semen horizontally on a 4 cm high foam board so that it floats on the liquid nitrogen surface; let it stand for 3 minutes, and then quickly transfer it to liquid nitrogen for storage.

[0038] (4) Thawing and resuscitation: After one month of cryopreservation, remove six sets of 2 mL cryovials containing semen from liquid nitrogen; quickly insert them into a 40°C water bath; shake continuously for about 30 seconds until the semen is in an ice-water mixture state; aspirate 1 μL of thawed semen onto a glass slide; adjust the microscope to the appropriate focus; add pure water to activate the sperm; observe sperm motility under a microscope; and use the sperm survival system (CASA) to test sperm survival. The results of the six groups of sperm survival tests are shown in Table 3. The sperm survival rate was 43.5 ± 6.8%. Figure 2 This is the thawed and revived longfin bream sperm observed in Comparative Example 1.

[0039] Table 3 shows the survival rate of 6 groups of longfin bream sperm after thawing and recovery in Comparative Example 1

[0040]

[0041] (5) Fertilization success rate experiment: The thawed and revived semen in step 5 was combined with female fish eggs under fertilization conditions. The fertilization rate was observed and recorded after 3 hours. The survival rate of the fry was observed and recorded after 3 weeks. The results of the fertilization success rate experiments of the six groups are shown in Table 4.

[0042] Table 4 is the experimental results of the fertilization success rate of 6 groups of long-finned bream sperm in comparative example 1

[0043]

[0044]

[0045] Comparative Example 2:

[0046] (1) Preparation of antifreeze solution: Pipette 7.5 mL of methanol into a 50 mL centrifuge tube, add trehalose, soybean lecithin and tea polyphenols at the same concentration as in Example 1 into the centrifuge tube; add Hank's solution standard solution diluent to 50 mL, and mix thoroughly with a vortex shaker to dissolve.

[0047] (2) Sperm collection: During the breeding season of longfin rayfish, cast a net to catch longfin rayfish males, select healthy males that are sexually mature, wipe the area around the cloaca with a dry towel, gently press the abdomen, squeeze the semen into a culture dish, and quickly transfer it to ice. Use a 10-μl pipette to draw 10 μl of semen into a 200-μl centrifuge tube, add 50 μl of Hank's solution standard diluent, and pipette to mix; draw 1 μl of the mixture onto a glass slide, adjust the microscope to the appropriate focus; add 10 μl of pure water diluent to activate the sperm, observe the sperm movement under a microscope, and use the sperm survival rate detection system (CASA) to detect the sperm survival rate. Select semen with a fresh sperm survival rate of more than 70% for cryopreservation.

[0048] (3) Sperm preservation: Use a pipette to transfer the semen into a 50 mL centrifuge tube and measure its volume at the same time; add antifreeze solution at a ratio of 1:1 and mix thoroughly by pipetting; measure 240 μL of the semen antifreeze solution mixture into a 2 mL cryovial, and after all the parts are divided, place them on ice for temporary storage; place the 2 mL cryovial containing semen horizontally on a 4 cm high foam board so that it floats on the liquid nitrogen surface; let it stand for 3 minutes, and then quickly transfer it to liquid nitrogen for storage.

[0049] (4) Thawing and resuscitation: After one month of cryopreservation, remove six sets of 2 mL cryovials containing semen from liquid nitrogen; quickly insert them into a 40°C water bath; shake continuously for about 30 seconds until the semen is in an ice-water mixture state; aspirate 1 μL of thawed semen onto a glass slide; adjust the microscope to the appropriate focus; add pure water to activate the sperm; observe sperm motility under a microscope; and use the sperm survival system (CASA) to test sperm survival. The results of the six groups of sperm survival tests are shown in Table 5, and the sperm survival rate reached 50.3±8.4%. Figure 3 This is the thawed and revived longfin bream sperm observed in Comparative Example 2.

[0050] Table 5 shows the survival rate of 6 groups of longfin bream sperm after thawing and recovery in Comparative Example 2

[0051]

[0052]

[0053] (5) Fertilization success rate experiment: The thawed and revived semen in step 5 was allowed to combine with female fish eggs under fertilization conditions. The fertilization rate was observed and recorded after 3 hours. The survival rate of the fry was observed and recorded after 3 weeks. The results of the fertilization success rate experiments of the six groups are shown in Table 6.

[0054] Table 6 is the experimental results of the fertilization success rate of 6 groups of long-finned bream sperm in comparative example 2

[0055] Comparison group number Fertilization rate (%) Fry survival rate (%) 1 58.4 59.7 2 60.2 57.3 3 57.9 54.6 4 53.6 51.8 5 58.5 56.1 6 52.1 52.0

Claims

1. A method for cryopreservation of sperm of longfin bream, characterized in that: The following steps are involved: Step 1. Construct a simulated aqueous solution for in vitro fertilization of longfin ray: add the following components to pure water: 16 g / L sodium chloride, 0.80 g / L potassium chloride, 2 g / L L-glucose, and 85.5 g / L trehalose. The water temperature is 16-20°C. Step 2. Fresh semen collection and survival rate testing: During the longfin ray breeding season, select healthy, sexually mature male fish, dry the area around the cloaca with a dry towel, gently press the abdomen, squeeze the semen into a culture dish, and quickly transfer it to ice; use a 10-μl pipette to draw 10 μl of semen into a 200-μl centrifuge tube; draw 1 μl of semen and add 60 μl of the simulated water solution prepared in step 1 to activate the sperm. After mixing, observe sperm motility under a microscope on a glass slide and test sperm survival using a sperm survival rate testing system. Select semen with a fresh sperm survival rate of more than 70% for cryopreservation; Step 3. Prepare a mixture of semen and antifreeze components: Use a pipette to transfer the diluted semen to a 50 mL centrifuge tube and measure its volume. Then, add the following liquid containing the following components in a 1:1 ratio by volume to the centrifuge tube: 0.096 g / L disodium hydrogen phosphate, 0.70 g / L sodium bicarbonate, 0.12 g / L potassium dihydrogen phosphate, 0.38 g / L calcium chloride dihydrate, 0.20 g / L magnesium chloride hexahydrate, 0.22 g / L magnesium sulfate heptahydrate, 16.5 g / L soybean lecithin, 150 mL / L methanol, and 300 μg / mL tea polyphenols. Mix thoroughly by pipetting and dissolve. Step 4. Aliquot: Place 240 μL of the mixture prepared in step 3 into each 2 mL cryovial. Once all aliquots are complete, store them on ice. Step 5: Cooling and freezing: Refrigerate the cryovials in a refrigerator at 4°C for 30 minutes, freeze at -20°C for 10 minutes, and freeze at -80°C for 12 minutes, then store in liquid nitrogen.

2. The method for cryopreservation of sperm of the longfin croaker according to claim 1, wherein: In step 3, the soybean lecithin is in powder form.

3. The method for cryopreservation of sperm of the longfin bream according to claim 1, characterized in that: In step 5, the cells were cooled to -80°C using a cell gradient cooling box and then stored in liquid nitrogen.

4. The method for cryopreservation of sperm of the longfin bream according to claim 1, characterized in that: When thawing the stored longfin ray sperm, remove the 2 mL cryovial containing semen from liquid nitrogen; quickly insert it into a 40°C water bath; shake it continuously until the semen is in an ice-water mixture state and thawed; after thawing, add the simulated water solution constructed in step 1 to activate the sperm.

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