Cryoprotectant solution and cryopreservation method for scallop sperm

Abstract

This invention discloses a cryoprotective solution and cryopreservation method for scallop sperm, relating to the field of aquaculture technology. The scallop sperm cryoprotective solution comprises dimethyl sulfoxide (DMSO) and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxy radicals, with sterile seawater as the solvent. The final volume percentage concentration of DMSO in the semen is 4%-8%, and the final concentration of 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxy radicals in the semen is 40-60 μM. This invention's scallop sperm cryoprotective solution and cryopreservation method solve the problems of high toxicity, complex procedures, and lack of dedicated solutions in existing scallop sperm cryoprotective solutions. It is mainly used for the cryopreservation of scallop sperm to achieve long-term preservation of germplasm resources.

Description

Technical Field

[0001] This invention relates to the field of aquaculture technology. More specifically, this invention relates to a cryoprotectant solution for scallop sperm and a method for cryopreservation. Background Technology

[0002] Comb scallop ( Chlamys farreri Shellfish are important marine economic shellfish with significant aquaculture value. However, their artificial cultivation has long faced technical bottlenecks such as germplasm degradation and difficulties in preserving superior families, necessitating the establishment of an efficient long-term germplasm resource preservation technology system.

[0003] Cryopreservation of sperm at extremely low temperatures is an effective means of long-term preservation of germplasm resources. While this technology is relatively mature in fish, research on it, especially in scallops, remains significantly insufficient. Existing techniques often directly borrow freezing methods from fish or other shellfish (such as oysters and abalone), failing to fully consider the unique biological characteristics of scallop sperm, leading to the following prominent shortcomings in practical applications:

[0004] 1. The cryoprotectant formulation is simple and highly toxic: Existing methods generally use a high concentration of a single cryoprotectant (such as DMSO), which can easily produce chemical toxicity to the sperm of the scallop, seriously affecting the sperm motility and fertilization ability after thawing.

[0005] 2. The freezing process is complex and impractical: Most existing solutions rely on programmed cooling devices to achieve the freezing process. Although the temperature control is precise, the equipment cost is high, the operation is cumbersome and time-consuming, making it difficult to promote and apply on a large scale in aquaculture sites.

[0006] 3. Lack of systematic optimization and dedicated solutions: Currently, there are no studies on the systematic optimization of key parameters (such as the type and concentration of cryopreservatives, equilibration time, etc.) for the cryopreservation of scallop sperm. Directly applying solutions from other species often results in unstable preservation effects due to biological differences, failing to meet the high fertilization rates required for germplasm resource preservation.

[0007] In summary, there is a lack of an ultra-low temperature cryopreservation scheme in the current technology that is tailored to the characteristics of scallop sperm, has low toxicity of cryoprotectants, and has a simple and efficient freezing procedure. Summary of the Invention

[0008] One object of the present invention is to provide a cryoprotectant solution for scallop sperm and a method for cryopreservation, so as to at least solve the above-mentioned problems.

[0009] To achieve the objectives and other advantages of this invention, a cryoprotectant solution for scallop sperm is provided, comprising dimethyl sulfoxide and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxo radicals, with sterile seawater as the solvent, wherein the final volume percentage concentration of dimethyl sulfoxide in the semen is 4%-8%, and the final concentration of 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxo radicals in the semen is 40-60 μM.

[0010] Preferably, the cryoprotectant solution for scallop sperm has a final volume percentage concentration of 6% for dimethyl sulfoxide in the semen and a final concentration of 50 μM for 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxy radical in the semen.

[0011] This invention also provides a method for cryopreservation of scallop sperm, comprising:

[0012] Step 1: Select scallop semen with sperm motility higher than 80% and temporarily store it in an environment of 0-4℃;

[0013] Step 2: Prepare the above-mentioned cryoprotectant solution for scallop sperm, wherein the volume percentage concentration of dimethyl sulfoxide is 8%-16% and the concentration of 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxy radical is 80-120 μM, and temporarily store it in an environment of 0-4℃.

[0014] Step 3: Mix the scallop semen with the scallop sperm cryoprotectant at a volume ratio of 1:1, and equilibrate at 0-4℃ for 5-10 minutes to obtain the semen protection solution mixture.

[0015] Step 4: Dispense the semen preservation solution mixture into straws, fumigate the straws at -80℃ to -100℃ for 8-12 minutes, and then immerse the straws in liquid nitrogen at -196℃ for preservation.

[0016] Preferably, the method for cryopreserving scallop sperm further includes step five: removing the cryopreserved straw from liquid nitrogen and immediately placing it in a seawater bath at 40±0.5℃ and shaking it until the ice crystals completely disappear to obtain thawed semen; mixing the thawed semen with an equal volume of equilibration solution A at 20℃ for 5-10 minutes, then mixing it with an equal volume of equilibration solution B at 20℃ for 5-10 minutes to obtain usable semen; wherein, equilibration solution A is a sterile seawater solution containing 2%-4% by volume of dimethyl sulfoxide and 20-30 μM of 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxo radicals, and equilibration solution B is a sterile seawater solution containing 0.5%-1% by volume of dimethyl sulfoxide and 10-15 μM of 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxo radicals.

[0017] Preferably, in the method for cryopreserving scallop sperm, in step three, the scallop sperm and scallop sperm cryoprotectant are first mixed at a volume ratio of 2:1 and equilibrated at 0-4℃ for 5 minutes. Then, an equal amount of scallop sperm cryoprotectant is added to achieve a volume ratio of 1:1. The mixture is then equilibrated at 0-4℃ for another 5 minutes to obtain a sperm preservation solution.

[0018] Preferably, in the method for cryopreserving scallop sperm, in step four, the fumigation temperature is controlled at -85±2℃ and the fumigation time is 10 minutes.

[0019] Preferably, in the method for cryopreservation of scallop sperm, the fumigation in step four is carried out in a liquid nitrogen vapor constant temperature fumigation device. The liquid nitrogen vapor constant temperature fumigation device includes: a double-layer container structure, which consists of an outer liquid nitrogen insulation chamber and an inner fumigation chamber, with a vacuum insulation layer between the inner and outer layers. The bottom of the liquid nitrogen insulation chamber is provided with a stainless steel liquid nitrogen inlet pipe communicating with the fumigation chamber. The fumigation chamber is equipped with a temperature sensor, a micro fan, and a porous support for supporting the straw. A temperature closed-loop control system includes a temperature controller and an electromagnetic shut-off valve located on the stainless steel liquid nitrogen inlet pipe. The signal input terminal of the temperature controller is connected to the temperature sensor, and the control output terminal is connected to the electromagnetic shut-off valve and the micro fan, respectively. It is used to replenish liquid nitrogen by controlling the opening and closing of the electromagnetic shut-off valve according to the real-time temperature in the fumigation chamber, and to maintain the continuous operation of the micro fan, so as to stabilize the fumigation temperature at -85±2℃.

[0020] The present invention has at least the following beneficial effects:

[0021] First, a cryoprotective solution for scallop sperm was prepared using 8%-16% dimethyl sulfoxide (DMSO) and 80-120 μM 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxy radicals, with sterile seawater as the solvent. DMSO acts as a permeable cryoprotectant, effectively reducing ice crystal formation and protecting cell membrane integrity. 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxy radicals act as antioxidants, effectively scavenging excess reactive oxygen species (ROS) generated during freezing, significantly reducing lipid peroxidation levels, maintaining the structural integrity of sperm cell membranes and acrosome membranes, and protecting mitochondrial function and key enzyme activity. The synergistic effect of both significantly reduces the damage to sperm cells caused by freezing, ensuring that sperm maintain high viability and function after thawing, providing a reliable foundation for the long-term preservation of scallop germplasm resources.

[0022] Secondly, a systematic method for cryopreservation of scallop sperm was provided through step-by-step control from sperm selection and preservation solution preparation to mixing, dispensing, and freezing. This method ensures stable sperm handling under low-temperature conditions and solves the problem of decreased sperm motility caused by temperature fluctuations and improper operation in traditional methods. Specifically, temporary storage and equilibration at 0-4℃ reduces sperm metabolic rate and minimizes pretreatment damage; fumigation followed by liquid nitrogen immersion after dispensing achieves gradual cooling, avoiding heat shock and rapid ice crystal formation. This cryopreservation method not only improves freezing efficiency but also ensures that sperm maintain high motility after long-term storage, providing practical and scalable technical support for artificial breeding and germplasm resource preservation.

[0023] Third, during the thawing process, the ice crystals were rapidly melted in a seawater bath at 40±0.5℃, avoiding recrystallization damage. Subsequently, the sperm were mixed with equilibration solutions A and B, gradually reducing the concentration of the cryoprotectant. This allowed the sperm to adapt to environmental changes, mitigated osmotic pressure shocks, and protected the integrity and function of the sperm membrane, significantly improving sperm motility and fertilization capacity after thawing. This thawing and equilibration process ensured that the cryopreserved sperm could be effectively used for artificial insemination, improving reproductive success rates.

[0024] Fourth, by mixing in steps, first mixing and balancing at a volume ratio of 2:1, and then adding an equal amount of cryoprotectant to achieve a 1:1 ratio, this stepwise mixing method allows sperm to gradually adapt to changes in the concentration of the cryoprotectant, effectively alleviating the sperm stress response caused by osmotic pressure mutations and improving the survival rate and stability of sperm before freezing.

[0025] Fifth, by setting the fumigation temperature at -85±2℃ and maintaining it for 10 minutes, this precise temperature control avoids the negative impact of temperature fluctuations, ensures the uniformity and repeatability of the freezing process, improves the success rate of cryopreservation, and enhances the functional recovery effect of sperm after thawing.

[0026] Sixth, a dedicated fumigation device is provided. Through a double-layer container structure and a closed-loop temperature control system, this device can automatically maintain the fumigation chamber temperature at -85±2℃, ensuring a uniform freezing environment. Temperature sensors and electromagnetic shut-off valves can adjust liquid nitrogen replenishment in real time, and a miniature fan further evens the temperature distribution, eliminating human error. This device improves the repeatability and efficiency of cryopreservation and significantly reduces sperm damage caused by temperature instability.

[0027] Seventh, using the cryopreservation method of the present invention, the fertilization rate of frozen and thawed scallop sperm is not significantly different from that of fresh sperm, proving that the cryopreservation method of the present invention can not only maintain the sperm's motility, but more importantly, completely protect its key biological fertilization function, providing a reliable guarantee for the practical application of germplasm resource preservation.

[0028] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0029] Figure 1 The graph shows the effects of different final concentrations of dimethyl sulfoxide, propylene glycol, and ethylene glycol on the fertilization rate of scallop sperm.

[0030] Figure 2 The figure shows the effect of cryopreservation with different final concentrations of dimethyl sulfoxide on the fertilization rate of scallop sperm.

[0031] Figure 3 The graph shows the effect of different thawing temperatures on the fertilization rate of fresh scallop sperm and sperm frozen and preserved with 6% dimethyl sulfoxide.

[0032] Figure 4 The graph shows the effect of different sperm-egg ratios on the fertilization rate of fresh scallop sperm and sperm cryopreserved with 6% dimethyl sulfoxide.

[0033] Figure 5 The graph shows the effect of glycine addition on the fertilization rate of frozen-thawed sperm in the scallop.

[0034] Figure 6 The graph shows the effect of glucose addition on the fertilization rate of frozen-thawed sperm in scallops.

[0035] Figure 7 The graph shows the effect of adding sucrose on the fertilization rate of frozen-thawed sperm in scallops.

[0036] Figure 8 The graph shows the effect of adding coenzyme Q10 on the fertilization rate of frozen-thawed sperm in the scallop.

[0037] Figure 9 The figure shows the effect of adding 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxygen radical (TEMPOL) on the fertilization rate of frozen-thawed sperm in the scallop.

[0038] Figure 10 This is a graph showing the effect of different sperm-egg ratios on the fertilization rate of frozen-thawed sperm in the scallop.

[0039] Figure 11 The graph shows the effects of different cryoprotectants on the sperm function indicators of frozen and thawed scallops.

[0040] Figure 12 The figure shows the effect of different cryoprotectants on the level of oxidative damage to sperm cells in the scallop after freeze-thaw.

[0041] Figure 13This is a graph showing the effect of different freezing and thawing methods on the fertilization rate of frozen-thawed sperm in scallops. Detailed Implementation

[0042] The present invention will now be described in further detail with reference to the embodiments and accompanying drawings, so that those skilled in the art can implement it based on the description.

[0043] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0044] It should be noted that, unless otherwise specified, the experimental methods described in the following embodiments are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified. In the description of this invention, the terms "lateral", "longitudinal", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", and "outer" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0045] Example 1: Effects of different cryoprotectants on sperm fertilization rate in scallop.

[0046] This embodiment aims to systematically screen and compare the toxic effects of three commonly used permeable cryoprotectants (dimethyl sulfoxide, propylene glycol, and ethylene glycol) on scallop sperm at different concentrations, in order to determine the type and concentration range of the cryoprotectant with the lowest toxicity and most suitable for subsequent cryopreservation.

[0047] (1) Semen collection and pretreatment: During the breeding season, healthy, sexually mature 2-year-old male scallops (weighing 29.5±5.7 g) were selected. They were air-dried at 19-23℃ for 60-120 minutes, then transferred to filtered seawater at 18-21℃ to induce ejaculation. Semen produced during the continuous ejaculation phase was collected, filtered through a 30-micron cell sieve, and temporarily stored at 0-4℃. Sperm motility was assessed using a microscope, and only semen with a motility greater than 80% was used for subsequent experiments.

[0048] (2) Preparation of multi-gradient cryoprotectant solutions: Using sterilized natural seawater filtered through 0.22 micrometers as solvent, a series of cryoprotectant solutions were prepared with volume fractions (v / v) of 8%, 12%, 16%, 20%, and 24% for dimethyl sulfoxide (DMSO), ethylene glycol (EG), and propylene glycol (PG), respectively, and pre-cooled at 0-4℃ for later use. When semen was mixed with the cryoprotectant solution at a volume ratio of 1:1, the final concentrations were 4%, 6%, 8%, 10%, and 12%. Filtered seawater without any cryoprotectant was used as a negative control group, following the same procedure as the experimental group.

[0049] (3) Equilibration and fertilization detection: Pre-cooled semen was mixed with different types and concentrations of protective solutions at a 1:1 volume ratio and equilibrated in an ice bath at 0-4℃ for 10 minutes. Subsequently, the equilibrated sperm suspension was directly mixed with fresh eggs at a sperm-to-egg ratio of 100:1 for fertilization experiments. After fertilization, excess sperm was washed away, and the fertilized eggs were placed in seawater at 20℃ to incubate until the morula stage, and the fertilization rate was calculated.

[0050] Experimental results are as follows Figure 1 As shown, all three preservatives exhibited significant concentration-dependent toxicity, meaning that sperm fertilization rate generally decreased with increasing concentration. At a concentration of 4%, only the dimethyl sulfoxide (DMSO) group showed no significant difference in fertilization rate compared to the control group. However, when the concentration increased to 10% and 12%, the fertilization rates of all preservative groups decreased significantly. The fertilization rates of ethylene glycol and propylene glycol at all concentration points were generally significantly lower than those of the DMSO group at the same concentration. Only the DMSO group showed higher fertilization rates at final concentrations of 6% and 8%.

[0051] Conclusion: This embodiment demonstrates that dimethyl sulfoxide (DMSO) is a superior cryoprotectant to ethylene glycol and propylene glycol for scallop sperm, with an optimal working concentration window of 6%-8%. This result establishes the core cryoprotectant type and basic concentration range for subsequent cryopreservation experiments.

[0052] Example 2: Effect of different final concentrations of dimethyl sulfoxide on the fertilization rate of cryopreserved scallop sperm.

[0053] Based on Example 1, this embodiment further verifies which dimethyl sulfoxide concentration can achieve the optimal post-freezing fertilization rate in a complete cryogenic freezing-thawing process.

[0054] (1) Semen collection: The method is the same as in Example 1. Semen with sperm motility >80% was selected. A portion of fresh semen was directly fertilized with an egg as a fresh control group.

[0055] (2) Preparation of cryoprotectant: Based on the conclusion of Example 1, dimethyl sulfoxide (DMSO) with final concentrations of 6% and 8% was selected for full-process cryoprotectant verification. Base solutions with corresponding DMSO volume fractions of 12% and 16% were prepared (so that they could be mixed 1:1 to achieve the target final concentration) and kept at 0-4℃ for later use.

[0056] (3) Freezing and thawing: Mix the semen and the preservation solution at a 1:1 volume ratio and equilibrate at 0-4℃ for 10 minutes. Then, dispense into 0.25 mL frozen straws and place them on a 6 cm thick foam board floating on liquid nitrogen (approximately -80 to -100℃). Fumigate for 10 minutes and finally immerse in liquid nitrogen (-196℃) for one week. To thaw, quickly place the straws in a 40℃ seawater bath and shake until completely thawed, then transfer them to 20℃ seawater for equilibration.

[0057] (4) Fertilization test: Fertilization test was performed immediately after thawing and equilibration, using the same method as in Example 1. Due to freezing damage caused during the freezing process, the sperm-egg ratio was changed to 10000:1.

[0058] Experimental results are as follows Figure 2 As shown, the experimental group using a final concentration of 6% dimethyl sulfoxide (DMSO) cryopreservation solution had a significantly higher fertilization rate of frozen-thawed sperm (48.49 ± 8.4%) than the group using 8% DMSO. This result clearly demonstrates that 6% is the optimal working concentration of DMSO throughout the entire cryopreservation process, providing sufficient cryoprotection while minimizing toxicity.

[0059] Example 3: Effect of different thawing temperatures on the fertilization rate of sperm from frozen scallops.

[0060] Based on Example 2, this embodiment further verifies the effect of different thawing temperatures on the fertilization rate of frozen scallop sperm in a complete cryogenic freezing-thawing process with the addition of 6% dimethyl sulfoxide, and determines the most suitable temperature for thawing frozen scallop sperm.

[0061] (1) Sperm collection: The method is the same as in Example 1. Semen with sperm motility >80% was selected. A portion of fresh semen was directly fertilized with an egg as a fresh control group.

[0062] (2) Preparation of cryoprotectant: Based on the conclusion of Example 2, a 12% dimethyl sulfoxide solution was prepared and stored at 0-4℃ for later use. When the semen and cryoprotectant were mixed at a volume ratio of 1:1, the final concentration was halved.

[0063] (3) Freezing and thawing: The method is the same as in Example 2. The semen and the protective solution are mixed at a ratio of 1:1, equilibrated at 0-4℃ for 10 minutes, dispensed, and then fumigated with liquid nitrogen (-80 to -100℃) for 10 minutes. The mixture is then stored in liquid nitrogen (-196℃) for one week. Thawing is performed by rapid recovery in seawater baths at 30℃, 40℃, 50℃, 60℃, and 70℃, and then transferred to seawater at 20℃ for equilibration.

[0064] (4) Fertilization test: Fertilization experiment was conducted immediately after thawing and equilibration. The fertilization method of the control group was the same as in Example 1. Fresh sperm suspension that had not been frozen was mixed with fresh eggs at a sperm-to-egg ratio of 100:1 for fertilization experiment (unless otherwise specified, the sperm-to-egg ratio of fresh sperm to eggs was always 100:1, the same below); the fertilization method of the experimental group was the same as in Example 2. Due to the freezing damage to sperm during the freezing process, all thawed sperm suspensions were mixed with fresh eggs at a sperm-to-egg ratio of 10000:1 for fertilization experiment.

[0065] Experimental results are as follows Figure 3 As shown, under 6% dimethyl sulfoxide conditions, the fertilization rate of frozen-thawed sperm at a thawing temperature of 40℃ (48.49±8.4%) was significantly higher than that at thawing temperatures of 30℃, 50℃, 60℃, and 70℃. This result indicates that 40℃ is the optimal thawing temperature for frozen-thawed sperm from *Scallop scallop* during the entire cryopreservation process, minimizing damage to the sperm during thawing.

[0066] Example 4: Effect of different sperm-egg ratios on the fertilization rate of frozen scallop sperm.

[0067] Based on Example 3, this embodiment further verifies the effect of the sperm-egg ratio on the fertilization rate of frozen and thawed sperm in a complete cryogenic freezing-thawing process with the addition of 6% dimethyl sulfoxide, and determines an evaluation condition that can sensitively distinguish the effects of different cryoprotectants.

[0068] (1) Sperm collection: The method is the same as in Example 1. Semen with sperm motility >80% was selected. A portion of fresh semen was directly fertilized with an egg as a fresh control group.

[0069] (2) Preparation of cryoprotectant: Based on the conclusion of Example 2, a 12% dimethyl sulfoxide solution was prepared and stored at 0-4℃ for later use. When the semen and cryoprotectant were mixed at a volume ratio of 1:1, the final concentration was halved.

[0070] (3) Freezing and thawing: The method is the same as in Example 3. The semen and the protective solution were mixed at a ratio of 1:1, equilibrated at 0-4°C for 10 minutes, dispensed, and then fumigated with liquid nitrogen (-80 to -100°C) for 10 minutes. The mixture was then stored in liquid nitrogen (-196°C) for one week. Based on the conclusion of Example 3, the semen was rapidly thawed using a 40°C seawater bath, and then transferred to 20°C seawater for equilibration.

[0071] (4) Fertilization test: Sperm from the 6% dimethyl sulfoxide freeze-thaw group were fertilized with fresh eggs at sperm-egg ratios of 10000:1, 20000:1, 30000:1 and 40000:1, and the fertilization rate during the morula stage was statistically analyzed.

[0072] Experimental results are as follows Figure 4 As shown, the fertilization rate of sperm in the 6% dimethyl sulfoxide (DMSO) freeze-thaw group gradually increased with the increase in the sperm-to-egg ratio. When the sperm-to-egg ratio reached 40,000:1, the fertilization rate of sperm in the 6% DMSO freeze-thaw group was no significantly different from that in the fresh sperm group. Therefore, 40,000:1 is the "critical point" for distinguishing the effect of the cryoprotectant in this experimental system. To screen cryoprotectants fairly and sensitively, subsequent experiments should be conducted at a sperm-to-egg ratio below or equal to this critical point to expose the true functional differences between frozen and thawed sperm.

[0073] Example 5: Effect of adding different antioxidants on the fertilization rate of sperm from frozen scallops.

[0074] Based on Example 4, this embodiment systematically screens antioxidants that can further improve the fertilization rate of frozen-thawed sperm at a sperm-egg ratio (40000:1) that can sensitively distinguish the protective effect.

[0075] (1) Sperm collection: The method is the same as in Example 1. Semen with sperm motility >80% was selected. A portion of fresh semen was directly fertilized with an egg as a fresh control group.

[0076] (2) Preparation of cryoprotectant: Based on the conclusions of Example 2, a final concentration of 6% dimethyl sulfoxide was selected as the positive control group, without the addition of any antioxidants, to participate in the freeze-thaw process and fertilize the eggs. Natural seawater filtered and sterilized at 0.22 μm was used as the solvent, and different types and concentrations of antioxidants were added to a 12% dimethyl sulfoxide solution. To highlight the comparative effect, this example focuses on listing the ineffective control and the effective ingredient. Examples of ineffective controls include, but are not limited to, glycosides.

[0077] Additives commonly used in sperm cryopreservation for other species include amino acids (0.4%-1.6%), glucose (0.5%-4%), sucrose (1%-8%), and coenzyme Q10 (10-80 μM). An effective example of this invention is 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxygen radical (TEMPOL), with a concentration gradient of 50-400 μM. The prepared cryoprotectant solution is stored at 0-4°C for later use. When semen and cryoprotectant solution are mixed at a volume ratio of 1:1, the final concentration is halved.

[0078] (3) Freezing and thawing: The method is the same as in Example 2. The semen and each protective solution are mixed at a ratio of 1:1, equilibrated at 0-4℃ for 10 minutes, dispensed, and then fumigated with liquid nitrogen (-80 to -100℃) for 10 minutes. The mixture is then stored in liquid nitrogen (-196℃) for one week. Thawing is performed by rapid recovery in a 40℃ seawater bath, followed by equilibration in 20℃ seawater.

[0079] (4) Fertilization test: After thawing and equilibration, sperm and fresh eggs were immediately mixed at a ratio of 40,000:1 for fertilization. The eggs were incubated in seawater at 20°C until the morula stage, and the fertilization rate was calculated.

[0080] Experimental results are as follows Figures 5 to 9 As shown, the fertilization rates of frozen-thawed sperm in all experimental groups supplemented with glycine, glucose, sucrose, and coenzyme Q10 were significantly different from those in the negative and positive control groups (6% dimethyl sulfoxide) using fresh sperm. Only the fertilization capacity of thawed sperm under the conditions of a final concentration of 6% dimethyl sulfoxide and 50 μM 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxygen radical (TEMPOL) showed no significant difference compared to the fertilization capacity of sperm under the conditions of fresh sperm control and positive control group (final concentration of 6% dimethyl sulfoxide). This demonstrates that TEMPOL has a specific and highly effective protective effect on the sperm of *Ctenophora indica*.

[0081] Example 6: Effect of sperm-egg ratio on sperm fertilization rate of scallops treated with different cryoprotectant solutions.

[0082] Based on Example 5, this embodiment verifies that after adding 50 μM TEMPOL, the sperm-to-egg ratio required for high fertilization rate of frozen-thawed sperm is significantly reduced, thus proving that the present invention can effectively improve the individual fertilization efficiency of frozen-thawed sperm, rather than simply relying on increasing the number of sperm to compensate for damage.

[0083] (1) Sperm collection: The method is the same as in Example 1. Semen with sperm motility >80% was selected. A portion of fresh semen was directly fertilized with fresh eggs as a fresh control group.

[0084] (2) Preparation of cryoprotectant: Based on the conclusions of Example 5, prepare a 12% dimethyl sulfoxide (DMSO) solution and a 12% DMSO + 100 μM TEMPOL solution. Store the prepared cryoprotectant at 0-4°C for later use. When the semen and cryoprotectant are mixed at a volume ratio of 1:1, a cryoprotectant solution with half the final concentration is achieved.

[0085] (3) Freezing and thawing: The method is the same as in Example 2. The semen and each protective solution are mixed at a ratio of 1:1, equilibrated at 0-4℃ for 10 minutes, dispensed, and then fumigated with liquid nitrogen (-80 to -100℃) for 10 minutes. The mixture is then stored in liquid nitrogen (-196℃) for one week. Thawing is performed by rapid recovery in a 40℃ seawater bath, followed by equilibration in 20℃ seawater.

[0086] (4) Fertilization test: After thawing and equilibration, sperm and fresh eggs were immediately mixed and fertilized at ratios of 10000:1, 20000:1, 30000:1, and 40000:1. The eggs were incubated in seawater at 20°C until the morula stage, and the fertilization rate was then calculated.

[0087] Experimental results are as follows Figure 9 As shown, under the conditions of a final concentration of 6% dimethyl sulfoxide and 50 μM 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxygen radical (TEMPOL), a sperm-to-egg ratio of 30,000:1 was achieved with a fertilization rate similar to the control group. This embodiment, through a sperm-to-egg ratio gradient experiment, confirms that compared to the scheme using only a final concentration of 6% dimethyl sulfoxide, the addition of 50 μM TEMPOL requires only a sperm-to-egg ratio of 30,000:1 to achieve a fertilization rate comparable to fresh sperm. The scheme without TEMPOL requires a sperm-to-egg ratio as high as 40,000:1. This demonstrates that the protective solution of this invention significantly improves the fertilization efficiency of frozen-thawed sperm, meaning that in practical germplasm resource preservation applications, the same amount of frozen sperm can be used to fertilize more eggs, greatly improving preservation efficiency and practicality.

[0088] Example 7: Effects of different treatments on sperm function indicators of frozen-thawed scallops.

[0089] Building upon Example 6, this embodiment explores the underlying mechanism by which the addition of 50 μM TEMPOL to the cryoprotectant significantly improves the fertilization rate of frozen-thawed sperm from the scallop *Ctenopharynx chinensis* at the cellular and molecular levels. By detecting key sperm functional indicators and oxidative stress levels, the hypothesis that TEMPOL protects sperm structure and function by mitigating oxidative damage is verified.

[0090] (1) Sperm collection: The method is the same as in Example 1. Semen with sperm motility >80% is selected.

[0091] (2) Preparation of cryoprotectant: Based on the conclusions of Example 6, a 12% dimethyl sulfoxide solution was prepared as the control group and a 12% dimethyl sulfoxide solution + 100 μM TEMPOL solution was prepared as the experimental group. The prepared cryoprotectant was stored at 0-4℃ for later use. When the semen and cryoprotectant were mixed at a volume ratio of 1:1, the final concentration was halved.

[0092] (3) Freezing and thawing: The method is the same as in Example 2. The semen and each protective solution are mixed at a ratio of 1:1, equilibrated at 0-4℃ for 10 minutes, dispensed, and then fumigated with liquid nitrogen (-80 to -100℃) for 10 minutes. The mixture is then stored in liquid nitrogen (-196℃) for one week. Thawing is performed by rapid recovery in a 40℃ seawater bath, followed by equilibration in 20℃ seawater.

[0093] (4) Sperm function indicators: Flow cytometry was used to detect multiple parameters in thawed sperm. Cell membrane integrity: Detected using the LIVE / DEAD sperm motility kit (SYBR-14 and propidium iodide (PI) double staining). Intact cell membranes repel PI and are stained green only by SYBR-14. Acrosome integrity: Detected using LysoTracker Green DD-26 (LYSO-G) and propidium iodide (PI) double staining. Intact acrosomes can be specifically labeled by LYSO-G. Mitochondrial membrane potential: Detected using Rhodamine 123 (Rh123) and propidium iodide (PI) double staining. The fluorescence intensity of Rh123 reflects the level of mitochondrial membrane potential and is an important indicator of mitochondrial function. The sperm density was adjusted to 5 × 10⁻⁶. 7 For each milliliter of sample, 1 milliliter was used for staining, and the staining conditions and concentrations were strictly performed according to the kit instructions and the methods described above. Flow cytometry was used for detection, and the percentage of positive cells was analyzed using FlowJo® v10 software.

[0094] (5) Detection of oxidative damage levels: Reactive oxygen species (ROS) level: detected using a reactive oxygen species detection kit. Lipid peroxidation level: detected using a lipid peroxidation detection kit. Specific procedures must be strictly followed according to the kit instructions.

[0095] Functional indicator results as follows Figure 11 As shown, compared with the control group (6% dimethyl sulfoxide), the sperm in the experimental group (6% dimethyl sulfoxide + 50 μM TEMPOL) had significantly higher cell membrane integrity, acrosome integrity, and mitochondrial membrane potential, which were closer to the levels of fresh sperm. The results of oxidative damage levels are as follows: Figure 12 As shown, compared with the control group (6% dimethyl sulfoxide), the sperm in the experimental group (6% dimethyl sulfoxide + 50 μM TEMPOL) had significantly lower levels of intracellular reactive oxygen species and lipid peroxidation products.

[0096] This embodiment demonstrates from a mechanistic perspective that the addition of 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxygen radical (TEMPOL) effectively reduces oxidative damage (manifested as a decrease in reactive oxygen species and lipid peroxidation levels) suffered by scallop sperm during ultra-low temperature freezing, thereby better maintaining the integrity of sperm cell membranes and acrosome structures, as well as mitochondrial function (manifested as an improvement in related indicators), ultimately ensuring that the thawed sperm possesses higher fertilization capacity. This provides a solid theoretical basis for the effectiveness of the cryoprotectant solution of this invention.

[0097] Example 8: Effects of different freezing and thawing methods and sperm-egg ratios on the fertilization rate of frozen-thawed sperm in scallops.

[0098] This embodiment aims to investigate the effects of different freeze-thaw methods on the fertilization rate of *Ctenopharynx scallop* sperm under different sperm-egg ratios, based on the cryoprotectant solution (final concentration 6% dimethyl sulfoxide and 50 μM 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxy radical) determined in Example 6. By comparing conventional freeze-thaw methods with the optimized freeze-thaw method of this invention, the significant advantages of the technical solution of this invention in improving the fertilization efficiency of frozen-thawed sperm are verified.

[0099] (1) Sperm collection: The method is the same as in Example 1. Semen with sperm motility >80% was selected. A portion of fresh semen was directly fertilized with fresh eggs as a fresh control group.

[0100] (2) Preparation of cryoprotectant: Based on the conclusions of Example 6, a cryoprotectant with a concentration of 12% dimethyl sulfoxide + 100 μM TEMPOL was prepared. The prepared cryoprotectant was stored at 0-4℃ for later use. When the semen and cryoprotectant were mixed at a volume ratio of 1:1, the final concentration of the cryoprotectant was halved.

[0101] (3) Freezing and Thawing: Two freezing and thawing methods were set up in the experiment. The positive control group used the method in Example 2: the semen and protective solution were mixed at a volume ratio of 1:1, equilibrated at 0-4℃ for 10 minutes, and then dispensed into 0.25 mL frozen straws. The frozen straws were placed on a 6 cm thick foam board floating on liquid nitrogen (about -80 to -100℃) and fumigated for 10 minutes. Then they were placed in liquid nitrogen (-196℃) for storage for one week. When thawing, the straws were quickly placed in a 40℃ seawater bath and shaken until completely thawed, and then transferred to 20℃ seawater for equilibration. The experimental group used the following method: the semen and protective solution were first mixed at a volume ratio of 2:1, equilibrated at 0-4℃ for 5 minutes, and then an equal amount of protective solution was added to make the volume ratio of semen to protective solution reach 1:1. The mixture was then equilibrated at 0-4℃ for 5 minutes, and then dispensed into 0.25 mL frozen straws. The frozen wheat tubes were inserted into a porous support of a liquid nitrogen vapor constant-temperature fumigation device at -85°C for 10 minutes. They were then stored in liquid nitrogen (-196°C) for one week. To thaw, the wheat tubes were quickly placed in a 40°C seawater bath and shaken until completely thawed. Then, at 20°C, the thawed semen was first mixed with an equal volume of equilibration solution A at 20°C and equilibrated for 5-10 minutes. Next, it was mixed with an equal volume of equilibration solution B at 20°C and equilibrated for 5-10 minutes. Equilibration solution A is a sterile seawater solution containing 3% dimethyl sulfoxide and 25 μM TEMPOL by volume, while equilibration solution B contains 0.75% dimethyl sulfoxide and 12.5 μM TEMPOL by volume. TEMPOL's sterile seawater solution; the liquid nitrogen vapor constant temperature fumigation device includes: a double-layer container structure, which consists of an outer liquid nitrogen insulation chamber and an inner fumigation chamber, with a vacuum insulation layer between the inner and outer layers. The bottom of the liquid nitrogen insulation chamber is equipped with a stainless steel liquid nitrogen inlet pipe that communicates with the fumigation chamber. The fumigation chamber is equipped with a temperature sensor, a miniature fan, and a porous support for supporting wheat straws; a temperature closed-loop control system, which includes a temperature controller and an electromagnetic shut-off valve located on the stainless steel liquid nitrogen inlet pipe. The signal input terminal of the temperature controller is connected to the temperature sensor, and the control output terminal is connected to the electromagnetic shut-off valve and the miniature fan, respectively. It is used to replenish liquid nitrogen by controlling the opening and closing of the electromagnetic shut-off valve according to the real-time temperature in the fumigation chamber, and to maintain the continuous operation of the miniature fan, so as to stabilize the fumigation temperature at -85±2℃.

[0102] (4) Fertilization test: After thawing and equilibration, sperm and fresh eggs were immediately mixed and fertilized at ratios of 15000:1, 20000:1, 25000:1, and 30000:1. The eggs were incubated in seawater at 20°C until the morula stage, and the fertilization rate was then calculated.

[0103] Experimental results are as follows Figure 13As shown, the positive control group only achieved a fertilization rate comparable to the fresh sperm control group at a sperm-to-egg ratio of 30,000:1, indicating no significant difference. In contrast, the experimental group demonstrated a significant advantage: at a ratio of 20,000:1, the fertilization rate was not statistically different from that of the fresh sperm control group.

[0104] The results of this experiment fully demonstrate that, using the same cryoprotectant solution, the optimized freeze-thaw method employed in this invention can significantly improve the fertilization efficiency of frozen-thawed sperm. Conventional methods require a sperm-to-egg ratio of 30,000:1 to achieve a fertilization rate comparable to fresh sperm, while the method of this invention only requires a ratio of 20,000:1, meaning a 33% saving in sperm usage in practical applications. This achievement of a high fertilization rate with a lower sperm-to-egg ratio overcomes the limitations of existing technologies, demonstrating the innovation and practicality of this invention, and providing more economical and efficient technical support for the preservation of *Scallop* germplasm resources.

[0105] The number of devices and processing scale described herein are for the purpose of simplifying the description of the invention. Applications, modifications, and variations of the scallop sperm cryoprotectant solution and cryopreservation method of the present invention will be readily apparent to those skilled in the art.

[0106] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A method for cryopreservation of sperm of Chlamys variegata, characterized by, The application relates to a sperm freezing method for Chlamys farreri. Step one: selecting Chlamys farreri sperm with a sperm activity higher than 80%, and temporarily storing the sperm in a 0-4 DEG C environment; Step two: preparing a Chlamys farreri sperm freezing protective solution, which comprises dimethyl sulfoxide and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, and the solvent is sterile seawater, wherein the volume percentage concentration of the dimethyl sulfoxide is 12%, and the concentration of the 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl is 100 muM, and the solution is temporarily stored in a 0-4 DEG C environment; Step three: firstly mixing the Chlamys farreri sperm and the Chlamys farreri sperm freezing protective solution according to a volume ratio of 2:1, balancing the mixture in a 0-4 DEG C environment for 5 minutes, then adding the same amount of the Chlamys farreri sperm freezing protective solution as the first time, so that the volume ratio of the Chlamys farreri sperm to the Chlamys farreri sperm freezing protective solution reaches 1:1, and balancing the mixture in a 0-4 DEG C environment for 5 minutes, to obtain a sperm protective solution mixed solution; Step four: distributing the sperm protective solution mixed solution into a macaroni tube, fumigating the macaroni tube in a-85+ / -2 DEG C environment for 10 minutes, and then storing the macaroni tube in liquid nitrogen at-196 DEG C; Step five: taking the frozen macaroni tube out of the liquid nitrogen, immediately placing the macaroni tube in a 40+ / -0.5 DEG C seawater bath and shaking the macaroni tube until the ice crystals completely disappear, to obtain thawed sperm; mixing the thawed sperm with an equal amount of 20 DEG C balanced liquid A, balancing for 5-10 minutes, mixing the thawed sperm with an equal amount of 20 DEG C balanced liquid B, balancing for 5-10 minutes, and obtaining available sperm; wherein the balanced liquid A is a sterile seawater solution containing 3% dimethyl sulfoxide and 25 muM 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl by volume percentage, and the balanced liquid B is a sterile seawater solution containing 0.75% dimethyl sulfoxide and 12.5 muM 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl by volume percentage.

2. The method of claim 1, wherein the sperm of the Chlamys hirunta is frozen and preserved by the method of claim 1, The fumigation in step four is carried out in a liquid nitrogen vapor constant-temperature fumigation device, and the liquid nitrogen vapor constant-temperature fumigation device comprises: a double-layer container structure which is composed of an outer layer liquid nitrogen heat preservation cavity and an inner layer fumigation cavity, a vacuum heat preservation layer is arranged between the inner layer and the outer layer, a stainless steel liquid nitrogen inlet pipe which is in communication with the fumigation cavity is arranged at the bottom of the liquid nitrogen heat preservation cavity, a temperature sensor, a micro fan and a porous support for loading the macaroni tube are arranged in the fumigation cavity; a temperature closed-loop control system which comprises a temperature controller and an electromagnetic shut-off valve arranged on the stainless steel liquid nitrogen inlet pipe, the signal input end of the temperature controller is connected with the temperature sensor, the control output end is connected with the electromagnetic shut-off valve and the micro fan respectively, and the temperature controller is used for supplementing liquid nitrogen by controlling the opening and closing of the electromagnetic shut-off valve and maintaining the continuous operation of the micro fan according to the real-time temperature in the fumigation cavity, so that the fumigation temperature is stably maintained at-85+ / -2 DEG C. ​

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