A method for cryopreservation of sperm of dwarf clams by vitrification

Abstract

The present invention discloses a method for cryopreservation of sperm of dwarf clams by vitrification, which relates to the field of marine biotechnology. The preservation method comprises the following steps: (1) taking dwarf clams in the breeding period, subjecting them to air-drying and temperature-changing stimulation in sequence to make them naturally discharge sperm, filtering and centrifuging to obtain high-density sperm, and storing it on ice; (2) mixing the high-density sperm obtained in step (1) with a cryoprotectant solution and placing it on ice to obtain a mixed sperm solution; the cryoprotectant solution is composed of dimethyl sulfoxide, coenzyme Q10 and polysucrose; (3) transferring the mixed sperm solution into a cryotube and directly immersing it in liquid nitrogen for storage. It has been found through research that after thawing at a temperature of 50-60 °C for 60-64 s, the D-shaped larva rate reaches more than 80%, realizing a cryopreservation method of directly putting the sample into liquid nitrogen for long-term storage.

Description

Technical Field

[0001] The present invention relates to the field of marine biotechnology, and particularly relates to a method for cryopreservation of dwarf surfclam sperm by vitrification. Background Art

[0002] Since the cryopreservation technology of sperm was first successfully developed in the 1950s last century, after more than half a century of development, it currently plays an important role in the protection of animal germplasm resources and genetic breeding research. At present, the traditional cryopreservation methods of sperm (non-programmed and programmed cryopreservation) have been widely used in aquaculture, including seawater bivalves such as oysters, mussels and scallops. However, this method requires the assistance of relevant freezing devices or instruments to complete the cryopreservation step at ultra-low temperature, which is time-consuming and laborious. The vitrification cryopreservation of sperm in this application is to directly put the sample into liquid nitrogen for cryopreservation after sample treatment, and the freezing process can be quickly completed without the assistance of freezing devices or instruments, which is more time-saving, labor-saving, convenient, simple and has lower production costs, and is convenient for popularization and application in genetic breeding production practice. At present, this method has not been reported in seawater bivalves.

[0003] In addition, for the economic seawater bivalves studied at present, due to their long breeding cycle and the fact that they grow at sea for most of the time and are difficult to manage, there are certain difficulties in the application and research of this method. Therefore, it is very important to select a suitable model organism for research. The dwarf surfclam has the characteristics of bivalves and has been used as a model organism for seawater bivalves. At present, taking the dwarf surfclam as the research object, the research results have been applied to economic seawater bivalves, such as Yang and Guo (Polyploid induction by heat shock-induced meiosis and mitosis inhibition in the dwarf surfclam, Mulinia lateralis Say. Aquaculture, 2006) developed a method for breeding polyploid seawater bivalves, and Wang et al. (Functional characterization of Cfap206 for bivalve ciliogenesis by RNAi and CRISPR / Cas9 technologies. Frontiers in Marine Science, 2022, 9: 864037) carried out research on the function of bivalve ciliogenesis using gene editing technology, and Guo et al. (Full-length transcriptome analysis provides insights into larval shell formation in Mulinia lateralis. In Frontiers in Marine Science, 2023, 9: 1111241), the research on the shell formation mechanism of bivalve larvae was carried out. At the same time, compared with other marine bivalves, the dwarf clam not only has the characteristics of a short sexual maturity cycle (about 3 months), but also the breeding methods and genomes of the entire life cycle of the dwarf clam have been established by this laboratory. Therefore, the dwarf clam is an ideal material for studying the vitrification cryopreservation of sperm of marine bivalves. At the same time, the applicant has developed a method for cryopreserving dwarf clam sperm by non-programmed cryopreservation. The vitrification cryopreservation method of the present invention is more time-saving, labor-saving and easy to standardize operation than the previous method on the premise of ensuring the sperm quality after freezing and thawing. Summary of the Invention

[0004] In view of the above-mentioned defects of the prior art, the present invention provides a method for vitrification cryopreservation of dwarf clam sperm to solve the problems raised in the above-mentioned background technology.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A method for vitrification cryopreservation of dwarf clam sperm, comprising the following steps:

[0007] (1) Take dwarf clams in the breeding period, and make them naturally discharge sperm by air-drying and temperature-changing stimulation in turn, filter, centrifuge, obtain high-density sperm, and store it on ice;

[0008] (2) Mix the high-density sperm obtained in step (1) with a cryoprotectant solution, and place it on ice to obtain a mixed sperm solution; the components of the cryoprotectant solution are composed of dimethyl sulfoxide, coenzyme Q10 and polysucrose;

[0009] (3) Transfer the mixed sperm solution into a cryotube and directly immerse it in liquid nitrogen for storage.

[0010] Preferably, in step (1), the air-drying is to air-dry the dwarf clams at 20-24°C for 60-90 minutes.

[0011] Preferably, in step (1), the temperature-changing stimulation is to place the dwarf clams in seawater at 26-27°C to naturally discharge sperm.

[0012] Preferably, the sperm filtration is carried out using a 24 µm sieve mesh.

[0013] Preferably, the density of the high-density sperm in step (1) is 1-1.5×10 8 cells / mL.

[0014] Preferably, the eggs required for the fertilization process are filtered using a 90 µm sieve mesh, directly collected and used directly without cryopreservation, and the density of the eggs is 1×10 4cells / mL.

[0015] Preferably, in step (2), the high-density sperm and the cryoprotectant are mixed at a volume ratio of 1:1.

[0016] Preferably, the time for placing on ice in step (2) is 15 - 20 min.

[0017] Preferably, the preparation method of the cryoprotectant in step (2) is to dissolve 16 mL of dimethyl sulfoxide, 0.003 g of coenzyme Q10, and 10 g of polysucrose in every 100 mL of filtered seawater, and stir evenly to obtain the cryoprotectant.

[0018] Preferably, the thawing method after the mixed sperm solution is immersed in liquid nitrogen for storage is to thaw in a water bath at 50 - 60 °C for 60 - 64 s.

[0019] Preferably, after thawing, the mixed sperm solution and the eggs are mixed for fertilization at a quantity ratio of 1000:1.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] (1) The vitrification cryopreservation method of dwarf clam sperm provided by the present invention, after thawing at a temperature of 50 - 60 °C for 60 - 64 s, it is found that this method helps to maintain the structural integrity and organelle functionality of sperm to the greatest extent after freeze-thawing, maintain good physiological and biochemical indexes. At the same time, it is found that the D-shaped larva rate reaches more than 80%, realizing a cryopreservation method that directly puts the sample into liquid nitrogen for long-term storage.

[0022] (2) In the existing method for cryopreserving shellfish sperm (Patent Application No.: CN202311267468.2), after adding the cryopreservation solution, the mixture of sperm and cryoprotectant is injected into a 0.25 mL cryotube, and then the cryotube is placed 4 cm above the liquid nitrogen surface for 10 minutes. After that, the cryotube is immersed in liquid nitrogen for long-term storage. In this process, a cryogenic device for storing liquid nitrogen needs to be purchased, and a 4 cm high rack is placed in the cryogenic device. Due to the limited length of the rack, the number of cryotubes that can be placed is limited (for example, if the 4 cm rack is 30 cm long, 20 - 25 cryotubes can be placed). In the experiment, before transferring into liquid nitrogen, the cryotubes are placed in the cryogenic device for cooling, which reduces the viability of the sperm samples. Moreover, the storage volume of each sample is only 0.25 mL, and the small storage volume of a single sample is not convenient for large-scale operations in the production practice of farms. In addition, the experimental process is complex, resulting in a reduction in the efficiency of cryopreserving samples. However, in this application, by appropriately selecting the components of the preservation solution, and currently only these three drug combinations can achieve vitrification cryopreservation, and this method has not been reported worldwide in shellfish. After sample treatment by this method, it can not only be directly placed in liquid nitrogen for long-term storage, but also belongs to a one-step cryopreservation method, without the need for a cryogenic device and a 4 cm rack for the pre-freezing process, avoiding the reduction of sperm sample viability and sperm structure damage caused by other external factors (before putting into liquid nitrogen, the more other experimental operation steps, the weaker the sperm viability and the stronger the damage degree of sperm structure); moreover, this method is not limited by the length of the high rack. As long as there are enough liquid nitrogen tanks, a large number of samples can be stored in the liquid nitrogen tanks. The experimental operation is simple and convenient, saving production costs and time, and can be standardized in production practice, thereby improving the viability and cryopreservation efficiency of sperm samples. This method can also store a large number of single samples. Currently, 2 mL cryotubes are used for storage, and the minimum storage volume of a single sample can be 0.4 mL, and the maximum can be 1.6 mL. It can store a large number of cryopreserved samples in the genetic breeding production practice, thereby improving the cryopreservation efficiency of the samples stored in production practice. Description of the Drawings

[0023] Figure 1 D-shaped larva rate (%) after thawing with different cryoprotectants, n = 3; different letters indicate significant differences;

[0024] Figure 2 D-shaped larva rate (%) after thawing with different concentrations of Ficoll as cryoprotectant, n = 3; different letters indicate significant differences;

[0025] Figure 3 D-shaped larva rate (%) after thawing with different sperm-egg ratios, n = 3; different letters indicate significant differences;

[0026] Figure 4The D-shaped larva rate (%) after freeze-thawing with different freezing volumes, n = 3. Different letters indicate significant differences;

[0027] Figure 5 Flow cytometry diagrams of fresh sperm and cryopreserved sperm of dwarf clams; among them, A is the cell membrane integrity; B is the mitochondrial membrane potential; C is the acrosome integrity. Specific implementation manners

[0028] To enable those skilled in the art to better understand the technical content of the present invention, the technical solutions of the present invention will be further described in detail below in conjunction with specific implementation manners.

[0029] Example 1

[0030] A method for cryopreserving dwarf clam sperm by vitrification and ultra-low temperature, comprising the following steps:

[0031] (1) Take 3-month-old dwarf clams with a weight of 0.26 ± 0.06 g. First, air-dry the sexually mature broodstock at room temperature for 60 min, then separate the male and female broodstock, and then place them in seawater at 26 °C for temperature change stimulation to naturally discharge sperm and eggs. The sperm is filtered using a 24-μm sieve mesh, and the fresh sperm with a vitality > 85% is centrifuged at 4 °C and 2000 g for 5 min to obtain 1×10 8 sperm / mL with a high density, and store it on ice; the eggs are filtered using a 90-μm sieve mesh and collected in a 24-μm sieve mesh to obtain 1×10 4 eggs / mL with a density, and store it at 20 °C;

[0032] (2) Mix the high-density sperm obtained in step (1) with the cryoprotectant in an equal volume ratio, and place it on ice for 15 min to obtain a mixed sperm solution; the preparation method of the cryoprotectant is to dissolve 16 mL of dimethyl sulfoxide, 0.003 g of coenzyme Q10, and 10 g of polysucrose in every 100 mL of filtered seawater, and stir evenly to obtain the cryoprotectant;

[0033] (3) Transfer the mixed sperm solution into a cryotube and directly immerse it in liquid nitrogen for storage.

[0034] Example 2

[0035] A method for cryopreserving dwarf clam sperm by vitrification and ultra-low temperature, comprising the following steps:

[0036] (1) Take 3-month-old dwarf clams with a weight of 0.26 ± 0.06 g. First, air-dry the sexually mature broodstock at room temperature for 90 min, then separate the male and female broodstock, and then place them in seawater at 27 °C for temperature change stimulation to naturally discharge sperm and eggs. The sperm is filtered using a 24-μm sieve mesh, and the fresh sperm with a vitality > 85% is centrifuged at 4 °C and 2000 g for 5 min to obtain 1.5×108 1×10 spermatozoa / mL of high-density spermatozoa were placed on ice for storage; egg filtration was carried out by filtering with a 90 µm silk mesh and collecting in a 24 µm silk mesh to obtain 1×10 4 eggs / mL of high-density eggs, which were placed at 24 °C for storage;

[0037] (2) The high-density spermatozoa obtained in step (1) were mixed with the cryoprotectant in an equal volume ratio and placed on ice for 20 min to obtain a mixed spermatozoa solution; the components of the cryoprotectant consisted of dimethyl sulfoxide, coenzyme Q10, and polysucrose; the preparation method of the cryoprotectant was to dissolve 16 mL of dimethyl sulfoxide, 0.003 g of coenzyme Q10, and 10 g of polysucrose in every 100 mL of filtered seawater, and stir evenly to obtain the cryoprotectant;

[0038] (3) The mixed spermatozoa solution was transferred into a cryotube and then directly immersed in liquid nitrogen for storage.

[0039] Experimental Example 1 Percentage of D-shaped larvae after thawing with different cryoprotectants

[0040] 1.1 Test method for the experimental group:

[0041] (1) Take dwarf clams at 3 months old with a body weight of 0.26 ± 0.06 g. The sexually mature broodstock were air-dried at room temperature for 60 - 90 min, and then the male and female broodstock were separated and placed in seawater at 26 - 27 °C to naturally discharge spermatozoa and eggs. The produced spermatozoa were filtered with a 24 µm silk mesh and then placed on ice for storage;

[0042] (2) Spermatozoa with fresh viability > 85% were centrifuged at 4 °C and 2000 g for 5 min to collect high-density spermatozoa, and the sperm density was set at 1×10 8 spermatozoa / mL and placed on ice for standby; fresh eggs were filtered with a 90 µm silk mesh to remove impurities and collected in a 24 µm silk mesh, and the egg density was set at 1×10 4 eggs / mL and placed at 20 - 24 °C for storage;

[0043] (3) Use a pipette to aspirate 0.2 mL each of high-density sperm and cryoprotectant solution, add them to a 2 mL cryotube and mix well to obtain a mixed sperm solution. Place it on ice for 20 min, and then directly transfer the 2 mL cryotube to liquid nitrogen for storage. The cryoprotectant solution has three components, namely, 16 mL of dimethyl sulfoxide and 0.003 g of coenzyme Q10 dissolved in every 100 mL of filtered seawater (Experimental Group 1: i.e., 8% DMSO + 20 μM Q10); 16 mL of dimethyl sulfoxide and 10 g of ficoll dissolved in every 100 mL of filtered seawater (Experimental Group 2: i.e., 8% DMSO + 146 mM FIC); 16 mL of dimethyl sulfoxide, 0.003 g of coenzyme Q10 and 10 g of ficoll dissolved in every 100 mL of filtered seawater (Experimental Group 3: i.e., 8% DMSO + 20 μM Q10 + 146 mM FIC).

[0044] (4) Thaw the mixed sperm solution stored in liquid nitrogen in a 60 °C water bath for 64 s, and then use the thawed mixed sperm solution for the experiment.

[0045] (5) Fertilize the thawed sperm and fresh eggs at a sperm-egg number ratio of 500:1. After 15 min, wash away the excess sperm with a 24 μm sieve mesh. Place the fertilized eggs in a 500 mL container and incubate them at 21 - 22 °C. Calculate the D-shaped larva rate after 24 h.

[0046] 1.2 Control group (control) test method: Artificially inseminate normal sperm (not cryopreserved) and eggs at a sperm-egg ratio of 5:1, then mix them evenly and incubate them in seawater at 21 - 22 °C. Calculate the D-shaped larva rate after 24 h.

[0047] 1.3 Matched group test method: Replace the ficoll in Experimental Group 3 with sucrose to prepare the cryoprotectant solution, and the remaining experimental steps are the same as those of the experimental group test method.

[0048] 1.4 Test results

[0049] As shown in Table 1:

[0050] Table 1 D-shaped larva rates after freezing and thawing with different cryoprotectants

[0051]

[0052] As can be seen from Table 1, compared with the control group, the D-shaped larva rate will decrease after cryopreservation, but the decrease rate in Experimental Group 3 is the lowest, indicating that the cryoprotectant prepared with 8% DMSO + 20 μM Q10 + 146 mM FIC can better maintain the viability of sperm and eggs; comparing Experimental Group 3 with the matched group, it can be seen that after replacing ficoll with sucrose, the D-shaped larva rate decreases significantly, indicating that the addition of ficoll in the cryoprotectant has a better preservation effect. The D-shaped larva rates of the control group and Experimental Groups 1 - 3 are shown in detail as followsFigure 1 as shown

[0053] Experimental Example 2 D-shaped larva rate after freeze-thawing with different concentrations of Ficoll as cryoprotectant

[0054] 2.1 Test method

[0055] (1)Take dwarf clams, 3 months old, with a weight of 0.26±0.06 g. Dry the sexually mature broodstock in the shade at room temperature for 60-90 min, then separate the male and female broodstock and place them in seawater at 26-27 °C to allow them to naturally release sperm and eggs. The released sperm is filtered through a 24-μm sieve mesh and then stored on ice;

[0056] (2)Centrifuge the sperm with a fresh vitality > 85% at 4 °C and 2000 g for 5 min to collect high-density sperm. Set the sperm density at 1×10 8 cells / mL and store it on ice for later use; Filter the fresh eggs through a 90-μm sieve mesh to remove impurities and collect them in a 24-μm sieve mesh. Set the egg density at 1×10 4 cells / mL and store them at 20-24 °C;

[0057] (3)Use a pipette to aspirate 0.2 mL of high-density sperm and cryoprotectant respectively, add them to a 2-mL cryotube and mix well to obtain a mixed sperm solution. Place it on ice for 20 min, and then directly transfer the 2-mL cryotube to liquid nitrogen for storage. There are 3 kinds of cryoprotectant components, which are 16 mL of dimethyl sulfoxide, 0.003 g of coenzyme Q10 and 5 g of Ficoll dissolved in every 100 mL of filtered seawater (Experimental Group 4, i.e., 8% DMSO + 20 μM Q10 + 73 mM FIC); 16 mL of dimethyl sulfoxide, 0.003 g of coenzyme Q10 (Experimental Group 5, i.e., 8% DMSO + 20 μM Q10 + 146 mM FIC) and 10 g of Ficoll dissolved in every 100 mL of filtered seawater; 16 mL of dimethyl sulfoxide, 0.003 g of coenzyme Q10 and 15 g of Ficoll dissolved in every 100 mL of filtered seawater (Experimental Group 6, i.e., 8% DMSO + 20 μM Q10 + 219 mM FIC);

[0058] (4)Thaw the mixed sperm solution stored in liquid nitrogen in a 60 °C water bath for 64 s, and then use the thawed mixed sperm solution for experiments;

[0059] (5)Fertilize the thawed sperm and fresh eggs at a sperm-egg number ratio of 500:1. After 15 min, wash away the excess sperm with a 24-μm sieve mesh. Place the fertilized eggs in a 500-mL container and incubate them at 21 °C. Calculate the D-shaped larva rate after 24 h.

[0060] 2.2 Control group test method: Normal sperm (not cryopreserved) and eggs were used for artificial insemination at a sperm-to-egg ratio of 5:1. After mixing, they were incubated in seawater at 21-22°C. After 24 hours, the D-larva rate was calculated.

[0061] 2.3 Test results

[0062] As shown in Table 2:

[0063] Table 2 D-larva rate after cryopreservation with different concentrations of Ficoll as cryoprotectant

[0064]

[0065] As shown in Table 2 and Figure 2 shown, the optimal concentration of Ficoll is 146 mM. Too high or too low a concentration of Ficoll can cause a decrease in the D-larva rate.

[0066] D-larva rate after fertilization with different sperm-to-egg ratios after cryopreservation in Experimental Example 3

[0067] 3.1 Test method

[0068] (1) Take dwarf clams, 3 months old, with a weight of 0.26 ± 0.06 g. The sexually mature broodstock were air-dried at room temperature for 60-90 minutes, and then the male and female broodstock were separated and placed in seawater at 26-27°C to allow them to naturally release sperm and eggs. The released sperm was filtered through a 24-μm sieve net and then stored on ice;

[0069] (2) The sperm with fresh motility > 85% was centrifuged at 4°C and 2000 g for 5 minutes to collect high-density sperm. The sperm density was set at 1×10 8 cells / mL and stored on ice; The fresh eggs were filtered through a 90-μm sieve net to remove impurities and collected in a 24-μm sieve net. The egg density was set at 1×10 4 cells / mL and stored at 20-24°C;

[0070] (3) Use a pipette to aspirate 0.2 mL of high-density sperm and cryoprotectant respectively, add them to a 2-mL cryotube and mix evenly to obtain a mixed sperm solution. Place it on ice for 20 minutes, and then directly transfer the 2-mL cryotube to liquid nitrogen for storage. The preparation method of the cryoprotectant is to dissolve 16 mL of dimethyl sulfoxide, 0.003 g of coenzyme Q10 and 10 g of Ficoll in every 100 mL of filtered seawater;

[0071] (4) The mixed sperm solution stored in liquid nitrogen was thawed in a water bath at 60°C for 64 s, and then the thawed mixed sperm solution was used for the experiment;

[0072] (5) Fertilize the thawed sperm and fresh eggs at sperm-egg number ratios of 500:1 (experimental group 7), 1000:1 (experimental group 8), and 1500:1 (experimental group 9). After 15 minutes, wash away the excess sperm with a 24-μm sieve silk net. Place the fertilized eggs in a 500-mL container and incubate at 21 - 22 °C. Calculate the D-shaped larva rate after 24 hours.

[0073] 3.2 Test method for the control group: Artificially inseminate normal sperm (not cryopreserved) and eggs at a sperm-egg ratio of 5:1, then mix well and incubate in seawater at 21 - 22 °C. Calculate the D-shaped larva rate after 24 hours.

[0074] 3.3 Test results

[0075] As shown in Table 3:

[0076] Table 3 D-shaped larva rates after fertilization with different sperm-egg ratios after freezing and thawing

[0077]

[0078] As shown in Table 3 and Figure 3 It can be seen that the best sperm-egg ratio is 1000:1. Too high or too low sperm-egg ratios can cause a decrease in the D-shaped larva rate.

[0079] Experimental Example 4 D-shaped larva rates after freezing and thawing with different freezing volumes

[0080] 4.1 Test method

[0081] (1) Take dwarf clams, 3 months old, with a weight of 0.26 ± 0.06 g. Air-dry the sexually mature parent clams at room temperature for 60 - 90 minutes, then separate the male and female parent clams and place them in seawater at 26 - 27 °C to allow them to naturally release sperm and eggs. Filter the released sperm with a 24-μm sieve silk net and then store it on ice.

[0082] (2) Centrifuge the fresh sperm with a motility > 85% at 4 °C and 2000 g for 5 minutes to collect high-density sperm. Set the sperm density at 1×10 8 cells / mL and store it on ice for later use; Filter the fresh eggs with a 90-μm sieve silk net to remove impurities and collect them in a 24-μm sieve silk net. Set the egg density at 1×10 4 cells / mL and store them at 20 - 24 °C.

[0083] (3) Use a pipette to aspirate 0.2 mL (experimental group 10), 0.4 mL (experimental group 11), and 0.8 mL (experimental group 12) of high-density sperm and cryoprotectant solution respectively, add them to a 2 mL cryotube and mix well to obtain a mixed sperm solution. Place it on ice for 20 min, and then directly transfer the 2 mL cryotube to liquid nitrogen for storage. The cryoprotectant solution is prepared by dissolving 16 mL of dimethyl sulfoxide, 0.003 g of coenzyme Q10, and 10 g of polysucrose in every 100 mL of filtered seawater;

[0084] (4) Thaw the mixed sperm solution stored in liquid nitrogen in a 60 °C water bath for 64 s, 95 s, and 115 s respectively, and then use the thawed mixed sperm solution for the experiment;

[0085] (5) Fertilize the thawed sperm and fresh eggs at a sperm-to-egg number ratio of 1000:1. After 15 min, wash away the excess sperm with a 24 µm sieve mesh, place the fertilized eggs in a 500 mL container, and incubate them at 21 - 22 °C. Calculate the D-shaped larva rate after 24 h.

[0086] 4.2 Test method for the control group: Artificially inseminate normal sperm (not cryopreserved) and eggs at a sperm-to-egg ratio of 5:1, then mix them evenly and incubate them in seawater at 21 - 22 °C. Calculate the D-shaped larva rate after 24 h.

[0087] 4.3 Test results

[0088] As shown in Table 4:

[0089] Table 4 D-shaped larva rate after freeze-thawing with different freezing volumes

[0090]

[0091] As shown in Table 4 and Figure 4 It can be seen that increasing the cryopreservation amount of each sample from 0.25 mL in the prior art to 1.6 mL greatly improves the cryopreservation efficiency.

[0092] Experimental Example 5 Sperm structural integrity and organelle functionality after freeze-thawing

[0093] 5.1 Test method

[0094] (1) Take dwarf clams, 3 months old, with a weight of 0.26 ± 0.06 g. Air-dry the sexually mature broodstock at room temperature for 60 - 90 min, then separate the male and female broodstock and place them in seawater at 26 - 27 °C respectively to allow them to naturally release sperm and eggs. The released sperm is filtered through a 24 µm sieve mesh and then stored on ice;

[0095] (2) Centrifuge sperm with fresh vitality > 85% at 4°C and 2000g for 5 min to collect high-density sperm. Set the sperm density at 1×10 8 cells / mL and place on ice for later use;

[0096] (3) Use a pipette to aspirate 0.4 mL each of high-density sperm and cryoprotectant solution, add them to a 2 mL cryotube, mix well, place on ice for 20 min, and then directly transfer the 2 mL cryotube to liquid nitrogen for storage. The cryoprotectant solution is prepared by dissolving 16 mL of dimethyl sulfoxide, 0.003 g of coenzyme Q10, and 10 g of polysucrose in every 100 mL of filtered seawater;

[0097] (4) Thaw the mixed sperm solution stored in liquid nitrogen in a 60°C water bath for 64 s, and then use the thawed mixed sperm solution for experiments.

[0098] Cell membrane integrity was detected using the LIVE / DEAD sperm viability kit: After freezing and thawing, dilute the sperm density to 2×10 6 cells / mL, take 1 mL of the diluted sperm for staining, add 100 μL of SYBR14 (4 μM) to the sperm and mix well. After 20 min, add 100 μL of propidium iodide (200 μM) and mix for 10 min;

[0099] Acrosome integrity was detected using the LysoTrack green DD-26 (LYSO-G) kit; Add 5 μL of LYSO-G (1000 μM) to the sperm and mix well. After 30 min, add 9 μL of propidium iodide (3000 μM) and mix for 10 min;

[0100] Mitochondrial membrane potential was detected using rhodamine 123 and propidium iodide: Add 100 μL of rhodamine 123 (10 μM) to the sperm and mix well. After 20 min, add 100 μL of propidium iodide (130 μM) and mix for 10 min;

[0101] The stained samples were observed using a flow cytometer (Beckman, CytoFLEX SRT, USA), and the survival rate was statistically analyzed.

[0102] 5.2 Test Results

[0103] The results are shown in Table 5:

[0104] Table 5 Comparison of cell membrane integrity, mitochondrial membrane potential, and acrosome integrity of sperm from fresh and frozen dwarf clams

[0105]

[0106] Note: Different letters indicate significant differences

[0107] Experimental Example 6 Determination of Physiological and Biochemical Indexes of Sperm after Freezing and Thawing

[0108] 6.1 Test Method

[0109] (1) Take dwarf clams, 3 months old, with a weight of 0.26 ± 0.06 g. Dry the sexually mature parent clams in the shade at room temperature for 60 - 90 min, then separate the male and female parent clams and place them in seawater at 26 - 27 °C respectively to allow them to naturally discharge sperm and eggs. The discharged sperm is filtered through a 24 - µm sieve mesh and then stored on ice;

[0110] (2) Centrifuge the fresh sperm with a motility > 85% at 4 °C and 2000 g for 5 min to collect high - density sperm, and set the sperm density at 1×10 8 cells / mL and store it on ice for later use;

[0111] (3) Use a pipette to aspirate 0.2 mL of high - density sperm and 0.2 mL of cryoprotectant respectively, add them to a 2 - mL cryotube, mix well, place it on ice for 20 min, and then directly transfer the 2 - mL cryotube to liquid nitrogen for storage. The cryoprotectant is prepared by dissolving 16 mL of dimethyl sulfoxide, 0.003 g of coenzyme Q10, and 10 g of polysucrose in every 100 mL of filtered seawater;

[0112] (4) Thaw the mixed sperm solution stored in liquid nitrogen in a water bath at 60 °C for 64 s, and then use the thawed mixed sperm solution for the experiment.

[0113] Superoxide dismutase is measured using a kit (EIASODC, Thermo Fisher Scientific, USA): Sperm (density 2.5×10 7 cells / mL) are centrifuged at 250 g for 10 min at 4 °C to obtain sperm. These sperm are mixed with pre - cooled PBS, sonicated for 1 min, and then centrifuged under the same conditions. The supernatant in the sample is further centrifuged at 1500 g for 10 min at 4 °C, and then the supernatant is transferred to another centrifuge tube. The superoxide dismutase activity of sperm is quantified using an enzyme - linked immunosorbent assay reader according to the instructions;

[0114] Catalase is measured using a kit (EIACATC, Thermo Fisher Scientific, USA): Sperm (density 2.5×10 7 cells / mL) are centrifuged at 250 g for 10 min at 4 °C to obtain sperm. These sperm are mixed with pre - cooled PBS, sonicated for 1 min, and then centrifuged under the same conditions. The supernatant in the sample is further centrifuged at 10000 g for 15 min at 4 °C, and then the supernatant is transferred to another centrifuge tube. The catalase activity of sperm is quantified using an enzyme - linked immunosorbent assay reader according to the instructions;

[0115] Glutathione enzyme kit (EIAGSHC, Thermo Fisher Scientific, USA) was used for determination: Sperm (density 2.5×10 7 cells / mL) was centrifuged at 250 g for 10 min at 4 °C to obtain sperm, and these sperm were mixed with pre-cooled PBS containing 5% 5-sulfosalicylic acid and incubated at 4 °C for 10 min. The sample was centrifuged at 18213 g for 10 min at 4 °C to obtain the supernatant, and the glutathione enzyme activity of sperm was quantified using a microplate reader according to the instructions.

[0116] 6.2 Test results

[0117] The results are shown in Table 6:

[0118] Table 6 Comparison of superoxide dismutase, catalase and glutathione enzyme activities of sperm of dwarf clams in the fresh and frozen groups

[0119]

[0120] Note: Different letters indicate significant differences

[0121] From the above results, although the structural integrity and enzyme activity of sperm of dwarf clams were significantly reduced after cryopreservation by vitrification, this method adopted a rapid cooling method. On the basis of the original dimethyl sulfoxide as the cryoprotectant, coenzyme Q10 and ficoll were added to achieve a cryopreservation method of directly putting the sample into liquid nitrogen for long-term storage. This method was successfully applied to marine bivalves for the first time. The freezing process was simple and easy to operate, and sperm cryopreservation at ultra-low temperature could be achieved without the aid of other freezing devices or instruments. At the same time, this method not only provided valuable reference for the development of cryopreservation technology of sperm of other economic clam species by vitrification at ultra-low temperature, but also laid a foundation for studying the impact of sperm cryopreservation at ultra-low temperature on the offspring of marine bivalves.

[0122] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for vitrification and ultra-low temperature cryopreservation of sperm of dwarf clams, characterized in that: The steps include: (1) taking dwarf clams in the reproductive period, drying them in the shade and stimulating them with temperature changes to allow them to naturally release sperm, filtering them, centrifuging them, obtaining high-density sperm, and storing them on ice; (2) mixing the high-density sperm obtained in step (1) with a cryoprotectant solution, and placing the mixture on ice to obtain a mixed sperm solution; the cryoprotectant solution comprises dimethyl sulfoxide, coenzyme Q10, and polysucrose; (3) Transfer the mixed sperm solution into a cryotube and directly immerse it in liquid nitrogen for storage; The preparation method of the cryoprotectant solution in step (2) is as follows: 16 mL of dimethyl sulfoxide, 0.003 g of coenzyme Q10 and 10 g of polysucrose are dissolved in every 100 mL of filtered seawater, and the mixture is stirred evenly to obtain the cryoprotectant solution.

2. The method for vitrification and ultra-low temperature cryopreservation of dwarf clam sperm according to claim 1, characterized in that: The shade drying in step (1) is to shade dry the dwarf clam at 20-24° C. for 60-90 minutes.

3. The method for vitrification and ultra-low temperature cryopreservation of sperm of dwarf clam according to claim 1, characterized in that: The temperature-changing stimulation in step (1) is to place the dwarf clam in seawater at 26-27° C. to naturally ejaculate sperm.

4. The method for vitrification and ultra-low temperature cryopreservation of sperm of dwarf clam according to claim 1, characterized in that: Sperm filtration was performed using a 24 μm mesh filter.

5. The method for vitrification and ultra-low temperature cryopreservation of dwarf clam sperm according to claim 1, characterized in that: The density of the high-density sperm in step (1) is 1 to 1.5×10 8 Pieces / mL.

6. The method for vitrification and ultra-low temperature cryopreservation of sperm of dwarf clam according to claim 1, characterized in that: In step (2), the high density sperm is mixed with the cryoprotectant solution in a volume ratio of 1:

1.

7. The method for vitrification and ultra-low temperature cryopreservation of sperm of dwarf clam according to claim 1, characterized in that: The time of placing on ice in step (2) is 15 to 20 minutes.

8. The method for vitrification and ultra-low temperature cryopreservation of dwarf clam sperm according to claim 1, characterized in that: The mixed sperm solution is thawed in a 50-60°C water bath for 60-64 seconds after being immersed in liquid nitrogen for storage.

9. The method for vitrification and ultra-low temperature cryopreservation of dwarf clam sperm according to claim 8, characterized in that: After thawing, the sperm solution and eggs are mixed in a ratio of 1000:1 for fertilization.

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