Vitrification ultra-low temperature cryopreservation method for dwarf sperms

Abstract

The invention discloses a dwarf sperm vitrification ultralow temperature cryopreservation method, and relates to the technical field of marine organisms. The preservation method comprises the following steps: (1) taking dwarf at a breeding period, sequentially drying in the shade and stimulating at variable temperature to naturally discharge sperms, filtering and centrifuging to obtain high-density sperms, and preserving the sperms on ice; (2) mixing the high-density sperms obtained in the step (1) with a cryoprotectant, and placing the mixture on ice to obtain a mixed sperm solution; the freezing protection liquid is prepared from the following components: dimethyl sulfoxide, coenzyme Q10 and polysucrose; and (3) transferring the mixed sperm solution into a freezing tube, and directly immersing the freezing tube into liquid nitrogen for preservation. Research finds that after the sample is unfrozen for 60-64s at the temperature of 50-60 DEG C, the rate of D-shaped larvae reaches 80% or above, and the freezing method that the sample is directly put into liquid nitrogen for long-term preservation is achieved.

Description

Technical Field

[0001] The invention relates to the field of marine biotechnology, and in particular to a method for vitrification and ultra-low temperature freezing and preservation of dwarf clam sperm. Background Art

[0002] Since the first success in the 1950s, sperm ultra-low temperature cryopreservation technology has developed for more than half a century and now plays an important role in animal germplasm resource protection and genetic breeding research. At present, traditional sperm ultra-low temperature cryopreservation methods (non-programmed and programmed ultra-low temperature freezing) have been widely used in aquaculture, including marine bivalve oysters, mussels and scallops, but this method requires the use of related freezing devices or instruments to complete the ultra-low temperature freezing step, which is time-consuming and labor-intensive. The sperm vitrification ultra-low temperature freezing of the present application is to directly put the sample into liquid nitrogen for cryopreservation after processing. The freezing process can be quickly completed without the help of a freezing device or instrument, which is more time-saving, labor-saving, convenient, simple and has a lower production cost, and is easy to promote and apply in genetic breeding production practice. At present, this method has not been reported in marine bivalve molluscs.

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

[0004] In view of the above-mentioned defects of the prior art, the present invention proposes a method for vitrification and ultra-low temperature cryopreservation of dwarf clam sperm to solve the problems raised by the above-mentioned background technology.

[0005] In order to achieve the above object, the present invention provides the following technical solutions: A method for vitrification and ultra-low temperature cryopreservation of sperm of dwarf clams comprises the following steps: (1) Take dwarf clams in the reproductive period, dry them in the shade and stimulate them to release sperm naturally, filter and centrifuge them to obtain high-density sperm, and store 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 freezing tube and directly immerse it in liquid nitrogen for storage.

[0006] Preferably, the shade drying in step (1) is to shade dry the dwarf clams at 20-24° C. for 60-90 min.

[0007] Preferably, the temperature-changing stimulation in step (1) is to place the dwarf clam in seawater at 26-27°C to allow it to naturally ejaculate sperm.

[0008] Preferably, sperm filtration is performed using a 24 µm mesh filtration.

[0009] Preferably, the density of the high-density sperm in step (1) is 1-1.5×10 8 Pieces / mL.

[0010] Preferably, the eggs used in the fertilization process are filtered through a 90µm silk screen and collected directly for use without being frozen. The density of the eggs is 1×10 4 Pieces / mL.

[0011] Preferably, in step (2), the high-density sperm and the cryoprotectant solution are mixed in a volume ratio of 1:1.

[0012] Preferably, the time of placing on ice in step (2) is 15 to 20 minutes.

[0013] Preferably, 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.

[0014] Preferably, the thawing method of the mixed sperm solution after being immersed in liquid nitrogen for storage is to thaw it in a 50~60ºC water bath for 60~64s.

[0015] Preferably, the thawed mixed sperm solution and eggs are mixed for fertilization in a ratio of 1000:1.

[0016] Compared with the prior art, the present invention has the following beneficial effects: (1) The vitrification ultra-low temperature cryopreservation method for dwarf clam sperm provided by the present invention, after thawing at a temperature of 50-60°C for 60-64 seconds, was found to help to maintain the structural integrity and organelle functionality of sperm to the maximum extent after freezing and thawing, and to maintain good physiological and biochemical indicators. At the same time, it was found that the D-shaped larvae rate reached more than 80%, realizing a freezing method in which the sample is directly placed in liquid nitrogen for long-term storage.

[0017] (2) In the prior art shellfish sperm cryopreservation method (patent application number: CN202311267468.2), after adding the cryopreservation solution, the mixture of sperm and cryoprotectant solution is injected into 0.25mL freezing straws, and then the freezing straws are placed 4cm away from the liquid nitrogen surface for 10min, and then immersed in liquid nitrogen for long-term storage. In this process, it is necessary to purchase a freezing device to hold liquid nitrogen, and a 4cm high shelf is placed in the freezing device. The shelf length is limited, and the number of freezing straws placed is limited (for example, a 4cm shelf is 30cm long, and 20-25 straws are placed). In the experiment, the straws were placed in the freezing device to cool down before being transferred to liquid nitrogen, which depleted the vitality of the sperm samples, and the storage volume of each sample was only 0.25mL. The storage volume of a single sample was small, which was not convenient for the farm to carry out large-scale operations in the production practice process, and the experimental process was complicated, resulting in a reduced efficiency of freezing samples. However, this application selects the appropriate components of the preservation solution, and currently only these three drug combinations can achieve vitrification freezing, and this method has not been reported in shellfish worldwide. After the sample is processed, this method can not only be directly placed in liquid nitrogen for long-term storage, but also belongs to a one-step freezing method, which does not require a freezing device or a 4cm shelf for pre-freezing, avoiding other external factors that cause sperm sample motility to decrease and sperm structure damage (before being placed in liquid nitrogen, the more other experimental operation steps, the weaker the sperm motility, and the stronger the degree of sperm structure damage); and this method is not limited by the height of the shelf length. As long as there are enough liquid nitrogen tanks, the samples can be stored in liquid nitrogen tanks in large quantities. The experimental operation is simple and convenient, saving production costs and time. Standardized operations can be performed in production practice, thereby improving sperm sample motility and cryopreservation efficiency. This method can also preserve a large number of single samples. Currently, 2ml cryovials are used for preservation. The minimum single sample can be stored at 0.4ml, and the maximum can be stored at 1.6ml. Frozen samples can be preserved in large quantities in genetic breeding production practice, thereby improving the cryopreservation efficiency of seed samples in production practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The D-shaped larvae rate (%) after freezing and thawing with different cryoprotectants, n=3; different letters indicate significant differences; Figure 2 The D-larvae rate (%) after freezing and thawing with different concentrations of polysucrose as cryoprotectant, n=3; different letters indicate significant differences; Figure 3 The D-shaped larvae rate (%) at different sperm-egg ratios after freezing and thawing, n=3; different letters indicate significant differences; Figure 4 The D-larvae rate (%) after freezing and thawing at different freezing volumes, n = 3. Different letters indicate significant differences; Figure 5 These are flow cytometric images of fresh and frozen sperm of dwarf clam; A represents cell membrane integrity; B represents mitochondrial membrane potential; and C represents acrosome integrity. DETAILED DESCRIPTION

[0019] In order to enable those skilled in the art to better understand the technical content of the present invention, the technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0020] Example 1 A method for vitrification and ultra-low temperature cryopreservation of sperm of dwarf clams comprises the following steps: (1) Three-month-old dwarf clams weighing 0.26±0.06 g were taken. The sexually mature clams were first dried in the shade at room temperature for 60 min. The male and female clams were then separated and placed in 26°C seawater for temperature stimulation to allow them to naturally release sperm and eggs. Sperm filtration was performed using a 24µm mesh. Fresh sperm with a motility of >85% were centrifuged at 4°C, 2000 g for 5 min to obtain 1×10 8 The sperm was stored on ice. The eggs were filtered through a 90-µm mesh and collected on a 24-µm mesh to obtain 1×10 4 Oocytes at a density of 100 / mL were stored at 20°C; (2) mixing the high-density sperm obtained in step (1) with a cryoprotectant solution in an equal volume ratio, and placing the mixture on ice for 15 minutes to obtain a mixed sperm solution; the cryoprotectant solution is prepared by dissolving 16 mL of dimethyl sulfoxide, 0.003 g of coenzyme Q10 and 10 g of polysucrose into 100 mL of filtered seawater, and stirring the mixture to obtain the cryoprotectant solution; (3) Transfer the mixed sperm solution into a freezing tube and directly immerse it in liquid nitrogen for storage.

[0021] Example 2 A method for vitrification and ultra-low temperature cryopreservation of sperm of dwarf clams comprises the following steps: (1) Three-month-old dwarf clams weighing 0.26±0.06 g were taken. The sexually mature clams were first dried in the shade at room temperature for 90 min. The male and female clams were then separated and placed in 27°C seawater for temperature stimulation to allow them to naturally release sperm and eggs. Sperm filtration was performed using a 24µm mesh. Fresh sperm with a motility of >85% were centrifuged at 4°C, 2000 g for 5 min to obtain 1.5×10 8 The sperm was stored on ice. The eggs were filtered through a 90-µm mesh and collected on a 24-µm mesh to obtain 1×10 4 Oocytes with high density of 100 / mL were stored at 24℃; (2) mixing the high-density sperm obtained in step (1) with a cryoprotectant in an equal volume ratio, and placing the mixture on ice for 20 minutes to obtain a mixed sperm solution; the cryoprotectant comprises dimethyl sulfoxide, coenzyme Q10 and polysucrose; the cryoprotectant is prepared by dissolving 16 mL of dimethyl sulfoxide, 0.003 g of coenzyme Q10 and 10 g of polysucrose into 100 mL of filtered seawater, and stirring the mixture to obtain the cryoprotectant; (3) Transfer the mixed sperm solution into a freezing tube and directly immerse it in liquid nitrogen for storage.

[0022] Experimental Example 1: D-larvae rate after freezing and thawing with different cryoprotectants 1.1 Experimental group test method: (1) Take dwarf clams, 3 months old, weighing 0.26±0.06g, and dry the sexually mature clams in the shade at room temperature for 60~90min. Then separate the male and female clams and place them in seawater at 26~27℃ to allow them to naturally ovulate sperm and eggs. The produced sperm is filtered with a 24µm silk mesh and then stored on ice; (2) Fresh sperm with motility > 85% were centrifuged at 4°C, 2000 g for 5 min to collect high-density sperm. The sperm density was set at 1 × 10 8 The fresh eggs were filtered through a 90-µm silk screen to remove impurities and collected in a 24-µm silk screen. The egg density was set at 1×10 4 / mL, stored at 20~24℃; (3) Use a pipette to draw 0.2 mL of high-density sperm and cryoprotectant solution, add them to a 2 mL cryotube and mix them evenly to obtain a mixed sperm solution. Place it on ice for 20 minutes, and then transfer the 2 mL cryotube directly to liquid nitrogen for storage. There are three types of cryoprotectant solution components: 16 mL of dimethyl sulfoxide and 0.003 g of coenzyme Q10 per 100 mL of filtered seawater (experimental group 1: 8% DMSO + 20 µM Q10); 16 mL of dimethyl sulfoxide and 10 g of polysucrose per 100 mL of filtered seawater (experimental group 2: 8% DMSO + 146 mM FIC); 16 mL of dimethyl sulfoxide, 0.003 g of coenzyme Q10 and 10 g of polysucrose per 100 mL of filtered seawater (experimental group 3: 8% DMSO + 20 µM Q10 + 146 mM FIC); (4) Thaw the mixed sperm solution stored in liquid nitrogen in a 60°C water bath for 64 seconds, and then use the thawed mixed sperm solution for the experiment; (5) Fertilize the thawed sperm with fresh eggs at a sperm-egg ratio of 500:1. After 15 minutes, wash away the excess sperm with a 24-µm silk mesh. Place the fertilized eggs in a 500-mL container and incubate at 21-22°C. Calculate the D-larvae rate after 24 hours.

[0023] 1.2 Control group test method: Artificial insemination of normal sperm (not frozen) and eggs at a sperm-egg ratio of 5:1, then mixed and incubated in seawater at 21-22°C, and the D-larvae rate was calculated after 24 hours.

[0024] 1.3 Matching group test method: replace the polysucrose in experimental group 3 with sucrose to prepare the cryoprotectant solution, and the rest of the experimental steps are the same as the experimental group test method.

[0025] 1.4 Test Results As shown in Table 1: Table 1 D-larvae rate after freezing and thawing with different cryoprotectants

[0026] As shown in Table 1, compared with the control group, the D-larvae rate decreased after freezing treatment, but the decrease rate in Experimental Group 3 was the lowest, indicating that the cryoprotectant prepared by 8% DMSO + 20µM Q10 + 146mM FIC can better maintain the vitality of sperm and eggs; compared with the matching group, it can be seen that after polysucrose was replaced with sucrose, the D-larvae rate decreased significantly, indicating that the preservation effect of adding polysucrose to the cryoprotectant is better. The D-larvae rate of the control group and Experimental Groups 1 to 3 is shown in the following figure. Figure 1 shown.

[0027] Experimental Example 2: D-larvae rate after freezing and thawing with different concentrations of polysucrose as cryoprotectant 2.1 Test Method (1) Take dwarf clams, 3 months old, weighing 0.26±0.06g, and dry the sexually mature clams in the shade at room temperature for 60~90min. Then separate the male and female clams and place them in seawater at 26~27℃ to allow them to naturally ovulate sperm and eggs. The produced sperm is filtered with a 24µm silk mesh and then stored on ice; (2) Fresh sperm with motility > 85% were centrifuged at 4°C, 2000 g for 5 min to collect high-density sperm. The sperm density was set at 1 × 10 8 The fresh eggs were filtered through a 90-µm silk screen to remove impurities and collected in a 24-µm silk screen. The egg density was set at 1×10 4 / mL, stored at 20~24℃; (3) Use a pipette to draw up 0.2 mL of high-density sperm and cryoprotectant solution, add them to a 2 mL cryotube and mix evenly to obtain a mixed sperm solution. Place it on ice for 20 min, and then transfer the 2 mL cryotube directly to liquid nitrogen for storage. There are three kinds of cryoprotectant solutions, namely, 16 mL of DMSO, 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 DMSO, 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 DMSO, 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); (4) Thaw the mixed sperm solution stored in liquid nitrogen in a 60°C water bath for 64 seconds, and then use the thawed mixed sperm solution for the experiment; (5) Fertilize the thawed sperm with fresh eggs at a sperm-egg ratio of 500:1. After 15 minutes, wash away the excess sperm with a 24-µm silk mesh. Place the fertilized eggs in a 500-mL container and incubate at 21°C. Calculate the D-larvae rate after 24 hours.

[0028] 2.2 Control group test method: Artificial insemination of normal sperm (not frozen) and eggs at a sperm-egg ratio of 5:1, then mixed and incubated in seawater at 21-22°C, and the D-larvae rate was calculated after 24 hours.

[0029] 2.3 Test Results As shown in Table 2: Table 2 Rate of D-shaped larvae after freezing and thawing with different concentrations of polysucrose as cryoprotectant

[0030] As shown in Table 2 and Figure 2 As shown, the optimal concentration of polysucrose is 146 mM. Too high or too low a concentration of polysucrose can cause a decrease in the rate of D-shaped larvae.

[0031] Experimental Example 3: D-shaped larvae rate after fertilization at different sperm-egg ratios after freezing and thawing 3.1 Test Method (1) Take dwarf clams, 3 months old, weighing 0.26±0.06g, and dry the sexually mature clams in the shade at room temperature for 60~90min. Then separate the male and female clams and place them in seawater at 26~27℃ to allow them to naturally ovulate sperm and eggs. The produced sperm is filtered with a 24µm silk mesh and then stored on ice; (2) Fresh sperm with motility > 85% were centrifuged at 4°C, 2000 g for 5 min to collect high-density sperm. The sperm density was set at 1 × 10 8 The fresh eggs were filtered through a 90-µm silk screen to remove impurities and collected in a 24-µm silk screen. The egg density was set at 1×10 4 / mL, stored at 20~24℃; (3) Use a pipette to draw 0.2 mL of high-density sperm and cryoprotectant solution, add them to a 2 mL cryotube and mix them evenly to obtain a mixed sperm solution. Place it on ice for 20 minutes, and then transfer the 2 mL cryotube directly 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; (4) Thaw the mixed sperm solution stored in liquid nitrogen in a water bath at 60°C for 64 seconds, and then use the thawed mixed sperm solution in the experiment; (5) Thawed sperm and fresh eggs were fertilized at a sperm-egg ratio of 500:1 (experimental group 7), 1000:1 (experimental group 8), and 1500:1 (experimental group 9). After 15 minutes, excess sperm was washed away with a 24-µm silk mesh. The fertilized eggs were placed in a 500-mL container and incubated at 21-22°C. The D-larvae rate was calculated after 24 hours.

[0032] 3.2 Control group test method: Artificial insemination of normal sperm (not frozen) and eggs at a sperm-egg ratio of 5:1, then mixed and incubated in seawater at 21-22°C, and the D-larvae rate was calculated after 24 hours.

[0033] 3.3 Test Results As shown in Table 3: Table 3 Rate of D-shaped larvae after fertilization at different sperm-egg ratios after freezing and thawing

[0034] As shown in Table 3 and Figure 3 It can be seen that the optimal sperm-egg ratio is 1000:1. Too high or too low a sperm-egg ratio can cause a decrease in the D-shaped larvae rate.

[0035] Experimental Example 4: D-larvae rate after freezing and thawing at different freezing volumes 4.1 Test Method (1) Take dwarf clams, 3 months old, weighing 0.26±0.06g, and dry the sexually mature clams in the shade at room temperature for 60~90min. Then separate the male and female clams and place them in seawater at 26~27℃ to allow them to naturally ovulate sperm and eggs. The produced sperm is filtered with a 24µm silk mesh and then stored on ice; (2) Fresh sperm with motility > 85% were centrifuged at 4°C, 2000 g for 5 min to collect high-density sperm. The sperm density was set at 1 × 10 8 The fresh eggs were filtered through a 90-µm silk screen to remove impurities and collected in a 24-µm silk screen. The egg density was set at 1×10 4 / mL, stored at 20~24℃; (3) Use a pipette to draw 0.2 mL (experimental group 10), 0.4 mL (experimental group 11), and 0.8 mL (experimental group 11) of high-density sperm and cryoprotectant solution, add them to a 2 mL cryotube and mix them evenly to obtain a mixed sperm solution. Place it on ice for 20 minutes, and then transfer the 2 mL cryotube directly 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; (4) Thaw the mixed sperm solution stored in liquid nitrogen in a 60°C water bath for 64s, 95s, and 115s, respectively, and then use the thawed mixed sperm solution in the experiment; (5) Fertilize the thawed sperm with fresh eggs at a sperm-egg ratio of 1000:1. After 15 minutes, wash away the excess sperm with a 24-µm silk mesh. Place the fertilized eggs in a 500-mL container and incubate at 21-22°C. Calculate the D-larvae rate after 24 hours.

[0036] 4.2 Control group test method: Artificial insemination of normal sperm (not frozen) and eggs at a sperm-egg ratio of 5:1, then mixed and incubated in seawater at 21-22°C, and the D-larvae rate was calculated after 24 hours.

[0037] 4.3 Test Results As shown in Table 4: Table 4 Rate of D-shaped larvae after freezing and thawing at different freezing volumes

[0038] As shown in Table 4 and Figure 4 It can be seen that increasing the cryopreservation volume of each sample from 0.25 mL in the prior art to 1.6 ml greatly improves the cryopreservation efficiency.

[0039] Experimental Example 5 Sperm structural integrity and organelle functionality after freezing and thawing 5.1 Test Method (1) Take dwarf clams, 3 months old, weighing 0.26±0.06g, and dry the sexually mature clams in the shade at room temperature for 60~90min. Then separate the male and female clams and place them in seawater at 26~27℃ to allow them to naturally ovulate sperm and eggs. The produced sperm is filtered with a 24µm silk mesh and then stored on ice; (2) Fresh sperm with motility > 85% were centrifuged at 4°C, 2000 g for 5 min to collect high-density sperm. The sperm density was set at 1 × 10 8 / mL, keep on ice for later use; (3) Use a pipette to draw 0.4 mL of high-density sperm and cryoprotectant solution, add them to a 2 mL cryotube and mix evenly, place on ice for 20 minutes, and then transfer the 2 mL cryotube directly 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; (4) Thaw the mixed sperm solution stored in liquid nitrogen in a 60°C water bath for 64 seconds, and then use the thawed mixed sperm solution for the experiment.

[0040] The cell membrane integrity was tested using the LIVE / DEAD sperm viability kit: the frozen-thawed sperm was diluted to a density of 2×10 6 / mL, take 1mL of diluted sperm for staining, take 100μL SYBR14 (4μM) and add it to the sperm and mix well. After 20min, add 100μL propidium iodide (200μM) and mix well for 10min; The acrosome integrity was tested using the LysoTrack green DD-26 (LYSO-G) kit; 5 μL LYSO-G (1000 μM) was added to the sperm and mixed well. After 30 min, 9 μL propidium iodide (3000 μM) was added and mixed well for 10 min. Mitochondrial membrane potential was detected using rhodamine 123 and propidium iodide: 100 μL of rhodamine 123 (10 μM) was added to sperm and mixed well. After 20 minutes, 100 μL of propidium iodide (130 μM) was added and mixed well for 10 minutes. The stained samples were observed by flow cytometer (Beckman, CytoFLEX SRT, USA) and the survival rate was calculated.

[0041] 5.2 Test Results The results are shown in Table 5: Table 5 Comparison of sperm cell membrane integrity, mitochondrial membrane potential and acrosome integrity in fresh and frozen dwarf clam groups

[0042] Note: Different letters indicate significant differences Experimental Example 6 Determination of Physiological and Biochemical Indexes of Sperm after Frozen-Thawed 6.1 Test Method (1) Take dwarf clams, 3 months old, weighing 0.26±0.06g, and dry the sexually mature clams in the shade at room temperature for 60~90min. Then separate the male and female clams and place them in seawater at 26~27℃ to allow them to naturally ovulate sperm and eggs. The produced sperm is filtered with a 24µm silk mesh and then stored on ice; (2) Fresh sperm with motility > 85% were centrifuged at 4°C, 2000 g for 5 min to collect high-density sperm. The sperm density was set at 1 × 10 8 / mL, keep on ice for later use; (3) Use a pipette to draw 0.2 mL of high-density sperm and cryoprotectant solution, add them to a 2 mL cryotube and mix evenly, place on ice for 20 minutes, and then transfer the 2 mL cryotube directly to liquid nitrogen for storage. The cryoprotectant solution is 16 mL of dimethyl sulfoxide, 0.003 g of coenzyme Q10, and 10 g of polysucrose dissolved in every 100 mL of filtered seawater; (4) The mixed sperm solution stored in liquid nitrogen was thawed in a water bath at 60°C for 64 seconds, and the thawed mixed sperm solution was then used in the experiment.

[0043] Superoxide dismutase was measured using a kit (EIASODC, Thermo Fisher Scientific, USA): sperm (density 2.5×10 7 cells / mL) were centrifuged at 250g for 10 min at 4°C to obtain sperm, which were then mixed with pre-cooled PBS, sonicated for 1 min, and centrifuged again under the same conditions. The supernatant in the sample was centrifuged at 1500g for 10 min at 4°C, and then placed in another centrifuge tube. The superoxide dismutase activity of sperm was quantified using an ELISA reader according to the instructions; Catalase was measured using a kit (EIACATC, Thermo Fisher Scientific, USA): sperm (density 2.5×10 7 cells / mL) were centrifuged at 250g for 10min at 4℃ to obtain sperm, which were then mixed with pre-cooled PBS, sonicated for 1min, and centrifuged again under the same conditions. The supernatant in the sample was centrifuged at 10000g for 15min at 4℃, and then placed in another centrifuge tube. The catalase activity of sperm was quantified using an ELISA reader according to the instructions; Glutathionease was measured using a kit (EIAGSHC, Thermo Fisher Scientific, USA): sperm (density 2.5×10 7cells / mL) at 250g for 10 min at 4°C to obtain sperm, which were mixed with pre-cooled PBS containing 5% 5-sulfosalicylic acid and incubated at 4°C for 10 min. The sample was centrifuged at 18213g for 10 min at 4°C to obtain the supernatant, and the sperm glutathionease activity was quantified using an ELISA reader according to the instructions.

[0044] 6.2 Test Results The results are shown in Table 6: Table 6 Comparison of superoxide dismutase, catalase and glutathionease activities in sperm of fresh and frozen dwarf clams

[0045] Note: Different letters indicate significant differences The above results show that although the structural integrity and enzyme activity of dwarf clam sperm are significantly reduced after vitrification cryopreservation, this method uses a rapid cooling method. On the basis of the original dimethyl sulfoxide as a cryoprotectant, coenzyme Q10 and polysucrose are added to achieve a freezing method that directly puts the sample into liquid nitrogen for long-term storage. This method was successfully used for the first time in marine bivalve molluscs. The freezing process is simple and easy to operate, and sperm can be ultra-low-temperature frozen without the help of other freezing devices or instruments. At the same time, this method not only provides a valuable reference for the development of sperm vitrification ultra-low-temperature cryopreservation technology for other economic clams, but also lays a foundation for studying the effects of sperm vitrification ultra-low-temperature cryopreservation on the offspring of marine bivalve molluscs.

[0046] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in 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) Take dwarf clams in the reproductive period, dry them in the shade and stimulate them to release sperm naturally, filter and centrifuge them to obtain high-density sperm, and store 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 clams 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 24µm 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 1-1.5×10 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 thawing method of the mixed sperm solution after being immersed in liquid nitrogen is to thaw it in a 50~60ºC water bath for 60~64s.

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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