Cryopreservation method of haliotis discus hannai sperms

Abstract

The invention discloses a cryopreservation method of haliotis diversicolor sperms. The method comprises the following steps: collecting the sperms, testing the initial activity of the sperms, preparing sperm freezing liquid, balancing and freezing the sperm freezing liquid, unfreezing the frozen sperms, and testing the activity after unfreezing. The collected male haliotis discus hannai sperms with the initial activity higher than 90% and a cryoprotectant are mixed according to the volume ratio of 1: 3, and sperm refrigerating fluid is obtained; transferring the sperm freezing liquid into straw, balancing, freezing, and soaking in liquid nitrogen for preservation to obtain frozen sperms; putting the straw filled with the frozen sperms into a water bath kettle, and unfreezing to obtain unfrozen sperms; finally, the straw containing the thawed sperms is cut open, the thawed sperms are transferred to a centrifugal tube, activity testing is carried out, and the activity and quality of the haliotis discus hannai sperms after cryopreservation are remarkably improved through the method.

Description

Technical Field

[0001] The invention relates to a method for freezing and preserving sperm of Haliotis discus hannai, and belongs to the ultra-low temperature freezing and preserving technology of animal sperm in cryobiology. Background Art

[0002] As a highly economically valuable shellfish, the hannai (Haliotis discus hannai) plays a crucial role in aquaculture. Its delicious, nutritious meat, rich in bioactive components such as polysaccharides and peptides, possesses antioxidant and anti-tumor properties. Consequently, it is highly sought after and commands a high price. With increasing demand for hannai, its cultivation has also expanded. However, in recent years, its aquaculture has faced numerous challenges, including low juvenile survival rates, slow growth, and increased mortality due to high temperatures. These factors have severely limited the production and economic benefits of abalone farming.

[0003] In the artificial breeding of abalone, in vitro fertilization requires a large amount of high-quality sperm. Genetically superior parent abalone are crucial for producing high-quality offspring, and sperm cryopreservation technology has become a key tool for obtaining high-quality parent abalone. Over the past decade, sperm cryopreservation technology for marine shellfish has garnered widespread attention, with numerous researchers conducting research on sperm cryopreservation techniques for various abalone species, including H. diversicolor, H. rufescens, and H. rubra.

[0004] Cryopreservation technology involves multiple steps, including semen collection, initial sperm quality assessment, cryoprotectant selection, cooling, thawing, and post-thaw sperm quality testing. Cryoprotectant formulation, equilibration time, and cooling rate are key factors influencing the success of cryopreservation. Currently, commonly used cryoprotectants include glycerol, dimethyl sulfoxide, methanol, ethylene glycol, and propylene glycol. However, different cryoprotectants exhibit varying protective effects on H. discus henna sperm, and a single cryoprotectant often fails to achieve optimal protection.

[0005] CN 101828546A discloses a method for cryopreservation of Haliotis discus hannai sperm. The method uses an antifreeze solution composed of glycerol, dimethyl sulfoxide, sucrose, and trehalose. Due to the complex composition of the antifreeze solution used in the method, the cost of cryopreservation is greatly increased. In order to reduce the cost of cryopreservation of Haliotis discus hannai sperm, the present invention provides a method for cryopreservation of Haliotis discus hannai sperm. The method reduces the cost by reducing the components of the refrigerant. Summary of the Invention

[0006] The present invention aims to optimize the cryopreservation scheme of Haliotis discus hannai sperm. By systematically studying the effects of different cryoprotectants (CPAs), equilibration time, freezing and thawing conditions, and other factors on the cryopreservation effect of sperm, the optimal cryoprotectant and its use conditions are determined, and the vitality of Haliotis discus hannai sperm after cryopreservation is improved, thereby providing technical support for large-scale seed production and germplasm resource conservation of Haliotis discus hannai.

[0007] In order to solve the problem that a single cryoprotectant cannot meet the demand for cryopreservation of the existing Haliotis discus hannai sperm, a method for cryopreservation of Haliotis discus hannai sperm is provided, the method comprising:

[0008] Sperm collection, initial sperm activity test, sperm freezing solution preparation, sperm freezing solution balance and freezing, thawing of frozen sperm, and activity test after thawing;

[0009] Sperm collection: Rinse the male Haliotis discus hannai with natural seawater, then use gauze to absorb the surface moisture of the washed abalone. Then use a sterile scalpel to cut open the abalone's adductor muscle to expose the viscera. Use a straw to aspirate the milky white semen to obtain the sperm of the male Haliotis discus hannai.

[0010] Initial sperm motility test: The collected male Haliotis discus hannai sperm was diluted with filtered seawater at a volume ratio of 1:200, retaining male Haliotis discus hannai sperm with an initial motility greater than 90%;

[0011] Preparation of sperm freezing solution: mixing the male Haliotis discus hannai sperm with an initial motility higher than 90% with a cryoprotectant in a volume ratio of 1:3 to obtain a sperm freezing solution;

[0012] Equilibration and storage of sperm freezing solution: transferring the sperm freezing solution into a straw, equilibrating, freezing, and storing in liquid nitrogen to obtain frozen sperm;

[0013] Thawing of frozen sperm: placing the straw containing the frozen sperm into a water bath for thawing to obtain thawed sperm;

[0014] Activity test after thawing: cut open the straw containing the thawed sperm, transfer the thawed sperm to a centrifuge tube, and perform activity test.

[0015] Optionally, the cryoprotectant comprises a permeable component and a non-permeable component;

[0016] The permeability component is selected from one of dimethyl sulfoxide, ethylene glycol, glycerol, and methanol with a final mass concentration of 10%;

[0017] The non-permeable component is selected from one of trehalose, glucose, sucrose, fructose, glycine and taurine with a final molar concentration of 0.04 mol / L.

[0018] Optionally, the cryoprotectant is dimethyl sulfoxide with a final mass concentration of 10% and trehalose with a final molar concentration of 0.04 mol / L.

[0019] Optionally, the equilibration time of the sperm freezing solution and the freezing is 0 to 2 hours;

[0020] The equilibrium temperature of the sperm freezing solution during equilibrium and freezing is 4°C.

[0021] Optionally, the equilibration time for the sperm freezing solution and freezing is 0.5 h.

[0022] Optionally, the freezing position during the sperm freezing solution equilibrium and freezing is 0 to 10 cm above the liquid nitrogen surface.

[0023] Optionally, the freezing position during sperm freezing liquid equilibrium and freezing is 5 cm above the liquid nitrogen surface.

[0024] Optionally, the sperm freezing liquid equilibration and freezing time is 10 minutes.

[0025] Optionally, the thawing temperature of the frozen sperm is 25-60°C.

[0026] Optionally, the thawing temperature of the frozen sperm is independently selected from any value among 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C or a range between any two of the above points.

[0027] Optionally, the thawing temperature of the frozen sperm is 50°C.

[0028] The technical solution disclosed in the present invention specifically includes the following parameters:

[0029] (1) Selection of cryoprotectants: Dimethyl sulfoxide (DMSO), ethylene glycol (EG), glycerol, methanol and trehalose (TRE), glucose (GLU), sucrose (SUC), fructose (FRU), glycine (GLY), and taurine (TAU) were selected as cryoprotectants to study the effects of different formulas of cryoprotectants on the cryopreservation of sperm of Abalone discus.

[0030] (2) Optimization of equilibration time: Four different equilibration times (0 h, 0.5 h, 1 h, 1.5 h, and 2 h) were set to explore the effect of equilibration time on sperm motility and determine the optimal equilibration time.

[0031] (3) Determination of freezing conditions: The freezing process is divided into two steps. First, a straw (0.25 mL) containing sperm is placed at different heights (0-10 cm) above the liquid nitrogen surface for 10 minutes. Then, the straw is quickly immersed in liquid nitrogen for storage. The effect of freezing conditions on sperm motility is studied to determine the optimal freezing conditions.

[0032] (4) Determination of thawing conditions: During thawing, the straws were thawed in a water bath at different temperatures (25°C, 40°C, 50°C and 60°C) to explore the effect of thawing temperature on sperm motility and determine the optimal thawing temperature.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] (1) Improving sperm cryopreservation: By optimizing various cryoprotectants and freezing conditions, the viability and quality of H. discus hannai sperm after cryopreservation were significantly improved. The results showed that the highest post-thaw sperm viability (57.24%) was achieved when 10% dimethyl sulfoxide and 0.04 mol / L trehalose were used as cryoprotectants, the equilibration time was 30 min at 4°C, the liquid nitrogen level was 5 cm during cryopreservation, and the thawing temperature was 50°C. This provides the optimal combination of cryoprotectants and conditions for the cryopreservation of H. discus hannai sperm.

[0035] (2) Providing technical support for seedling production: The optimal cryoprotectant and freezing storage conditions determined by the present invention can effectively preserve the vitality of the sperm of the wrinkled abalone, providing a reliable technical guarantee for the in vitro fertilization and seedling production of the wrinkled abalone, helping to improve the seedling yield and quality of the wrinkled abalone and promote the development of the wrinkled abalone aquaculture industry.

[0036] (3) Germplasm conservation: The optimization of sperm cryopreservation technology provides a feasible method for the long-term preservation of H. discus houndii germplasm resources. By cryopreserving the sperm of high-quality broodstock, the genetic diversity of H. discus houndii can be effectively protected, providing an important resource reserve for dealing with possible germplasm degeneration and disease threats in the future.

[0037] (4) The technical solution disclosed in the present application uses a combination of permeable dimethyl sulfoxide and non-permeable trehalose. Permeable dimethyl sulfoxide has the effects of lowering the freezing point of intracellular fluid, making the ice crystals formed during cryopreservation small and uniform, and regulating the osmotic pressure inside and outside the cells. Non-permeable trehalose can prevent cells from being directly damaged by ice crystals, interact with lipids and proteins on the cell membrane, stabilize the cell membrane structure and function, and stabilize the extracellular matrix. When the two work together, they can enhance the cell protection effect, stabilize the cell membrane structure, and improve the stability of bioactive substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a graph showing the test results of the effects of different cryoprotectants on sperm motility in Example 1 of the present invention (cryoprotective solutions were obtained by mixing four permeating cryoprotectants with six non-permeating cryoprotectants, with permeating cryoprotectant A being dimethyl sulfoxide; permeating cryoprotectant B being ethylene glycol; permeating cryoprotectant C being methanol; and permeating cryoprotectant D being glycerol);

[0039] Figure 2 This is a graph showing the test results of the effect of different equilibrium times on sperm motility in Example 2 of the present invention;

[0040] Figure 3 This is a graph showing the test results of Example 3 of the present invention showing the effect of different static liquid nitrogen levels on sperm motility;

[0041] Figure 4 This is a graph showing the test results of the effect of different thawing temperatures on sperm motility in Example 4 of the present invention;

[0042] Figure 5 This is a graph showing the sperm motility test results after thawing when using a permeable cryoprotectant alone in Comparative Example 1 of the present invention. DETAILED DESCRIPTION

[0043] The present invention will be further described below with reference to specific examples, but the present invention is not limited thereto in any way.

[0044] The samples of Haliotis discus hannai used in the embodiment of the present invention were collected in the Bohai Sea during its breeding season (May-June). The collected samples were high-quality male abalone of Haliotis discus hannai that were over 3 years old, had large and swollen gonads, and were milky white in color (male). They were transported to the laboratory at low temperature and then raised in a laboratory culture system for 7 days at a culture temperature of 16.5°C.

[0045] Dimethyl sulfoxide (DMSO), ethylene glycol (EG), glycerol, methanol, trehalose (TRE), glucose (GLU), sucrose (SUC), fructose (FRU), glycine (GLY), and taurine (TAU) used in the embodiments of the present invention are all from Sigma-Aldrich Co., Ltd. (USA); the natural seawater used is filtered seawater (FSW) obtained by passing through a 0.22 μm water filter membrane.

[0046] The freezing system of the present invention consists of a polystyrene foam box (35 cm long x 35 cm wide x 35 cm high) filled with liquid nitrogen and an aluminum mesh for containing the sample to be frozen.

[0047] The present invention is based on a technical solution that at least includes the following: sperm collection, sperm initial activity test, sperm freezing solution preparation, sperm freezing solution balance and freezing, frozen sperm thawing and activity recovery.

[0048] 1. Sperm collection: Rinse the male discus discus with natural seawater, then use gauze to absorb the surface moisture of the washed abalone. Then use a sterile scalpel to cut the adductor muscle of the abalone to expose its viscera. Use a straw to absorb the milky white semen, place it in a bottle and store it in a refrigerator at 4℃. To reduce experimental errors, sperm was collected from three different male abalone.

[0049] 2. Initial sperm motility test: The sperm solution collected in step 1 was diluted with filtered seawater at a volume ratio of 1:200. Sperm motility was assessed using an IVSO II instrument (Hamilton-Thorne, USA) and computer-assisted sperm analysis. Sperm samples with an initial sperm motility greater than 90% were selected for subsequent experiments.

[0050] 3. Preparation of sperm freezing solution: Mix the sperm collected in step 1 (activity above 90%) with cryoprotectant at a volume ratio of 1:3 and place in a straw.

[0051] 4. Equilibration and freezing of sperm freezing solution: Place the straw containing sperm freezing solution in step 3 in a refrigerator at 4°C for equilibrium. After equilibrium, place it in a freezing system for freezing to obtain frozen sperm.

[0052] 5. Thawing and motility test of frozen sperm: Place the straw containing the frozen sperm obtained in step 4 in a water bath and thaw to obtain the thawed semen; cut the straw and transfer the thawed semen to a centrifuge tube for motility test.

[0053] Example 1

[0054] Different cryoprotectants and sperm were cryopreserved to verify the effect of cryoprotectant composition on sperm activity after thawing; four permeability components (A: dimethyl sulfoxide, B: ethylene glycol, C: methanol, and D: glycerol) at a final mass concentration of 10% were mixed with six non-permeability components (Trehalose, sucrose, fructose, glucose, glycine, and taurine) at a final molar concentration of 0.04 mol / L to obtain cryoprotectants, using filtered seawater as the solvent; 62.5 μL of sperm (with activity greater than 90%) were mixed with 187.5 μL of the above cryoprotectants to obtain 250 μL of sperm cryopreservation solution, which was then placed in a straw and equilibrated in a refrigerator at 4°C for 0.5 hour. After equilibration, the straw containing the sperm cryopreservation solution was placed 3 cm above the liquid nitrogen level in the cooling system for 10 minutes, and finally stored in liquid nitrogen;

[0055] Place the straw containing frozen sperm in a 25°C water bath and thaw for 15 seconds to obtain the thawed semen;

[0056] Cut open the straw containing the thawed semen and transfer the thawed semen to a centrifuge tube for sperm motility recovery. The sperm motility test results after three parallel experiments in each group are as follows: Figure 1 As shown (A, B, C, and D represent dimethyl sulfoxide, ethylene glycol, methanol, and glycerol, respectively), the test results show that when the final mass concentration of the permeability cryoprotectant is 10% dimethyl sulfoxide and the final molar concentration of the non-permeability component is 0.04 mol / L trehalose as the cryoprotectant, the sperm of the wrinkled abalone has the highest vitality after thawing.

[0057] Example 2

[0058] The difference from Example 1 is that different equilibration times are selected to verify their effects on sperm motility after thawing. In this example, 10% dimethyl sulfoxide and 0.04 mol / L trehalose are selected as cryoprotectants, and 62.5 μL of sperm (activity higher than 90%) are mixed with 187.5 μL of 10% dimethyl sulfoxide (final concentration) and 0.04 mol / L trehalose (final concentration) to obtain a sperm freezing solution. The freezing solution is then placed in a straw and equilibrated in a refrigerator at 4°C for 0 h, 0.5 h, 1 h, 1.5 h, and 2 h, respectively. The other conditions are the same as in Example 1. The results of the activity test after three parallel experiments in each group are as follows: Figure 2 As shown, the test results show that when 10% dimethyl sulfoxide (final concentration) and 0.04 mol / L trehalose (final concentration) are selected as cryoprotectants, the freezing solution is balanced at 4°C for 0.5h (i.e. 30min), the sperm motility of the wrinkled abalone is significantly higher than that of other groups of experiments, and the sperm motility tends to decrease with the increase of equilibrium time.

[0059] Example 3

[0060] The difference from Examples 1 and 2 is that different liquid nitrogen levels were selected to verify their effects on sperm activity after thawing. In this example, 10% dimethyl sulfoxide (final concentration) and 0.04 mol / L trehalose (final concentration) were used as cryoprotectants. After the semen and cryoprotectant were mixed, they were equilibrated in a refrigerator at 4°C for 0.5 h. The sperm freezing solution was placed at different heights above the liquid nitrogen surface (0 cm, 3 cm, 5 cm, 8 cm, and 10 cm) for 10 min. The samples were then immersed in liquid nitrogen for storage. The specific test results of sperm activity after thawing are shown in FIG. Figure 3 As shown (three parallel experiments for each group), the results showed that the sperm recovery motility when the sperm freezing solution was placed 3cm and 5cm above the liquid nitrogen surface was significantly higher than that at other liquid level heights (P<0.05), and the sperm motility obtained at 5cm was higher (51.27%).

[0061] Example 4

[0062] The difference from Examples 1 to 3 is that different thawing temperatures are used to affect the activity of sperm after thawing; in this example, 10% dimethyl sulfoxide (final concentration) and 0.04 mol / L trehalose (final concentration) are selected as cryoprotectants, and the semen and cryoprotectant are mixed and equilibrated in a refrigerator at 4°C for 0.5 h. The sperm freezing solution is placed 5 cm above the liquid nitrogen surface for 10 min, and the straws containing frozen sperm are placed in water baths at 25°C, 40°C, 50°C and 60°C, respectively, and thawed for 15 seconds to obtain thawed semen; the straws containing thawed semen are cut open, and the thawed semen is transferred to a centrifuge tube for sperm motility recovery. The sperm activity test results after recovery are as follows: Figure 4 As shown (three parallel experiments for each group), the results showed that when 10% dimethyl sulfoxide and 0.04 mol / L trehalose were used as cryoprotectants, the equilibrium time was 0.5 h, and the sperm was placed 5 cm above the liquid nitrogen surface during cryopreservation, and the thawing temperature was 50°C, the sperm motility was the highest at 57.24%, which was significantly higher than other thawing temperatures.

[0063] Comparative Example 1

[0064] The difference from Examples 1 to 4 is that the cryoprotectant used is one of dimethyl sulfoxide, ethylene glycol, glycerol, and methanol (the sperm motility after thawing when the permeable cryoprotectant is used alone is the same). The other conditions are consistent with those of Example 1. The sperm motility test results after thawing are as follows: Figure 5 As shown, the results showed that when the cryoprotectant was dimethyl sulfoxide, the activity of sperm after thawing was 46.77%; when the cryoprotectant was ethylene glycol, the activity of sperm after thawing was 31.47%; when the cryoprotectant was glycerol, the activity of sperm after thawing was 41.47%; when the cryoprotectant was methanol, the activity of sperm after thawing was 32.43%.

[0065] The above descriptions are merely several embodiments of the present invention and do not constitute any form of limitation to the present invention. Although the present invention is disclosed as above in terms of preferred embodiments, they are not intended to limit the present invention. Any technician familiar with the present profession who, without departing from the scope of the technical solution of the present invention, makes slight changes or modifications using the technical contents disclosed above are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. A method for freezing and preserving sperm of Haliotis discus, characterized in that: The method comprises: Sperm collection, initial sperm activity test, sperm freezing solution preparation, sperm freezing solution balance and freezing, thawing of frozen sperm, and activity test after thawing; Sperm collection: Rinse the male Haliotis discus hannai with natural seawater, then use gauze to absorb the surface moisture of the washed abalone. Then, use a sterile scalpel to cut open the abalone's adductor muscle to expose the viscera. Use a straw to aspirate the milky white semen and place it in a bottle to obtain the male Haliotis discus hannai sperm. Initial sperm motility test: The collected male Haliotis discus hannai sperm was diluted with filtered seawater at a volume ratio of 1:200, retaining male Haliotis discus hannai sperm with an initial motility greater than 90%; Preparation of sperm freezing solution: mixing the male Haliotis discus hannai sperm with an initial motility higher than 90% with a cryoprotectant in a volume ratio of 1:3 to obtain a sperm freezing solution; Equilibration and freezing of sperm freezing solution: transferring the sperm freezing solution into a straw, equilibration, freezing, and immersing in liquid nitrogen for storage to obtain frozen sperm; Thawing of frozen sperm: placing the straw containing the frozen sperm into a water bath for thawing to obtain thawed sperm; Activity test after thawing: cut open the straw containing the thawed sperm, transfer the thawed sperm to a centrifuge tube, and perform activity test.

2. The cryopreservation method according to claim 1, wherein The cryoprotectant includes a permeable component and a non-permeable component; The permeability component is selected from one of dimethyl sulfoxide, ethylene glycol, glycerol, and methanol with a final mass concentration of 10%; The non-permeable component is selected from one of trehalose, glucose, sucrose, fructose, glycine and taurine with a final molar concentration of 0.04 mol / L.

3. The cryopreservation method according to claim 2, wherein The cryoprotectant is dimethyl sulfoxide with a final mass concentration of 10% and trehalose with a final molar concentration of 0.04 mol / L.

4. The cryopreservation method according to claim 1, wherein The equilibration time of the sperm freezing solution and the freezing is 0 to 2 hours; The equilibrium temperature of the sperm freezing solution during equilibrium and freezing is 4°C; Preferably, the equilibration time of the sperm freezing solution and the equilibration time during freezing is 0.5 h.

5. The cryopreservation method according to claim 1, wherein The freezing position during the sperm freezing solution balance and freezing is 0 to 10 cm above the liquid nitrogen surface; The freezing time during the sperm freezing solution equilibration and freezing is 10 minutes; Preferably, the freezing position during the sperm freezing liquid equilibrium and freezing is 5 cm above the liquid nitrogen surface.

6. The cryopreservation method according to claim 1, wherein The thawing temperature of the frozen sperm is 25-60°C; Preferably, the thawing temperature of the frozen sperm is 50°C.

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